U.S. patent application number 12/854449 was filed with the patent office on 2012-02-16 for aquaculture feed compositions.
This patent application is currently assigned to E. I. DU PONT DE NEMOURS AND COMPANY. Invention is credited to Scott E. Nichols, J. Martin Odom.
Application Number | 20120040076 12/854449 |
Document ID | / |
Family ID | 44515039 |
Filed Date | 2012-02-16 |
United States Patent
Application |
20120040076 |
Kind Code |
A1 |
Nichols; Scott E. ; et
al. |
February 16, 2012 |
AQUACULTURE FEED COMPOSITIONS
Abstract
Aquaculture feed compositions having a ratio of concentration of
.omega.-3 polyunsaturated fatty acids of eicosapentaenoic acid
["EPA"; cis-5,8,11,14,17-eicosapentaenoic acid; 20:5] to
concentration of docosahexaenoic acid ["DHA";
cis-4,7,10,13,16,19-docosahexaenoic acid; 22:6] greater than 2:1
based on the individual concentrations of EPA and DHA in the
aquaculture feed composition are disclosed. Furthermore, such
aquaculture feed compositions may have a concentration of the sum
of EPA and DHA that is at least about 0.8%, measured as a weight
percent of the aquaculture feed composition.
Inventors: |
Nichols; Scott E.; (West
Chester, PA) ; Odom; J. Martin; (Kennett Square,
PA) |
Assignee: |
E. I. DU PONT DE NEMOURS AND
COMPANY
WILMINGTON
DE
|
Family ID: |
44515039 |
Appl. No.: |
12/854449 |
Filed: |
August 11, 2010 |
Current U.S.
Class: |
426/601 |
Current CPC
Class: |
A23K 10/22 20160501;
Y02A 40/818 20180101; A23K 20/158 20160501; A23K 20/179 20160501;
A23K 50/80 20160501 |
Class at
Publication: |
426/601 |
International
Class: |
A23K 1/18 20060101
A23K001/18; A23K 1/10 20060101 A23K001/10 |
Claims
1. An aquaculture feed composition comprising: (a) at least one
source of eicosapentaenoic acid ["EPA"] and optionally at least one
source of docosahexanoic acid ["DHA"], wherein said source can be
the same or different; and, (b) a ratio of concentration of EPA to
concentration of DHA which is greater than 2:1 based on the
individual concentrations of EPA and DHA in the aquaculture feed
composition.
2. The aquaculture feed composition of claim 1 wherein said
composition further comprises a total amount of EPA and DHA that is
at least about 0.8%, measured as a weight percent of the
aquaculture feed composition.
3. The aquaculture feed composition of either claim 1 or 2 wherein
the at least one source of EPA is a first source that is microbial
oil and an optional second source that is fish oil or fish
meal.
4. The aquaculture feed composition of either claim 1 or 2 wherein
the at least one source of DHA is selected from the group
consisting of: microbial oil, fish oil, fish meal, and combinations
thereof.
5. The aquaculture feed composition of claim 3 wherein the
microbial oil is provided in a form selected from the group
consisting of: biomass, processed biomass, partially purified oil
and purified oil, any of which is obtained from at least one
transgenic microbe engineered for the production of polyunsaturated
fatty acid-containing microbial oil comprising EPA.
6. The aquaculture feed composition of claim 5 wherein the at least
one transgenic microbe is cultured.
7. The aquaculture feed composition of claim 5 wherein the biomass,
processed biomass, partially purified oil and/or purified oil are
obtained from the cultured transgenic microbe.
8. The aquaculture feed composition of claim 5 wherein the
transgenic microbe is Yarrowia lipolytica.
9. The aquaculture feed composition of claim 6 wherein the
transgenic microbe is Yarrowia lipolytica.
10. The aquaculture feed composition of claim 7 wherein the
transgenic microbe is Yarrowia lipolytica.
11. The aquaculture feed composition of claim 1 further comprising
vegetable oil.
12. A method of making an aquaculture feed composition comprising:
a) providing at least one source of eicosapentaenoic acid ["EPA"]
and optionally at least one source of docosahexanoic acid ["DHA"],
wherein said source can be the same or different; b) providing
additional feed components; c) contacting (a) and (b) to make an
aquaculture feed composition; wherein said aquaculture feed
composition has a ratio of concentration of EPA to concentration of
DHA which is greater than 2:1 based on the individual
concentrations of EPA and DHA in the aquaculture feed
composition.
13. The method of claim 12 further wherein said composition
comprises a total amount of EPA and DHA that is at least about
0.8%, measured as weight percent of the aquaculture feed
composition.
14. The method of either of claim 12 or 13 wherein the at least one
source of EPA is a first source that is microbial oil and an
optional second source that is fish oil or fish meal.
15. The method of either of claim 12 or 13 wherein the at least one
source of DHA is selected from the group consisting of: microbial
oil, fish oil, fish meal, and combinations thereof.
16. The method of claim 14 wherein the microbial oil is provided in
a form selected from the group consisting of biomass, processed
biomass, partially purified oil and purified oil, any of which is
obtained from at least one transgenic microbe engineered for the
production of polyunsaturated fatty acid-containing microbial oil
comprising EPA.
17. The method of claim 14 wherein the transgenic microbe is
Yarrowia lipolytica.
18. The method of claim 16 wherein the transgenic microbe is
Yarrowia lipolytica.
Description
FIELD OF THE INVENTION
[0001] This invention is in the field of aquaculture. More
specifically, this invention pertains to aquaculture feed
compositions with omega-3 polyunsaturated fatty acid ratios of
eicosapentaenoic acid to docosahexaenoic acid that are higher than
currently available using fish oil.
BACKGROUND OF THE INVENTION
[0002] Aquaculture is a form of agriculture that involves the
propagation, cultivation and marketing of aquatic animals and
plants in a controlled environment. The history of aquaculture in
the United States can be traced back to the mid to late 19.sup.th
century, when pioneers began to supply brood fish, fingerlings and
lessons in fish husbandry to would-be aquaculturists. Until the
early 1960's, commercial fish culture in the United States was
mainly restricted to rainbow trout, bait fish and a few warmwater
species (e.g., buffaloes, bass and crappies).
[0003] The aquaculture industry is currently the fastest growing
food production sector in the world. World aquaculture produces
approximately 60 million tons of seafood, which is worth more than
$70 billion (US) annually. Today, farmed fish accounts for
approximately 50% of all fish consumed globally. This percentage is
expected to increase, as a result of dwindling catches from capture
fisheries in both marine and freshwater environments and increasing
seafood consumption (i.e., total and per capita). Today, species
groups in aquaculture production include, for example: carps and
other cyprinids; oysters; clams, cockles and arkshells; shrimps amd
prawns; salmons, trouts and smelts; mussels; tilapias and other
cichlids; and scallops and pectens.
[0004] While some aquacultured species (e.g., Tilapia) can be fed
on an entirely vegetarian diet, many others species are fed a
carnivorous diet. Typically, the feed for carnivorous fish
comprises fishmeal and fish oil derived from wild caught species of
small pelagic fish (predominantly anchovy, jack mackerel, blue
whiting, capelin, sandeel and menhaden). These pelagic fish are
processed into fishmeal and fish oil, with the final product often
being either a pelleted or flaked feed, depending on the size of
the fish (e.g., fry, juveniles, adults). The other components of
the aquaculture feed composition may include vegetable protein,
vitamins, minerals and pigment as required.
[0005] Marine fish oils have traditionally been used as the sole
dietary lipid source in commercial fish feed given their ready
availability, competitive price and the abundance of essential
fatty acids contained within this product. Additionally, fish oils
readily supply essential fatty acids which are required for regular
growth, health, reproduction and bodily functions within fish. More
specifically, all vertebrate species, including fish, have a
dietary requirement for both omega-6 and omega-3 polyunsaturated
fatty acids ["PUFAs"]. Eicosapentaenoic acid ["EPA";
cis-5,8,11,14,17-eicosapentaenoic acid; .omega.-3] and
docosahexaenoic acid ["DHA"; cis-4,7,10,13,16,19-docosahexaenoic
acid; 22:6 .omega.-3] are required for fish growth and health and
are often incorporated into commercial fish feeds via addition of
fish oils.
[0006] It is estimated that aquaculture feed compositions currently
use about 87% of the global supply of fish oil as a lipid source.
Since annual fish oil production has not increased beyond 1.5
million tons per year, the rapidly growing aquaculture industry
cannot continue to rely on finite stocks of marine pelagic fish as
a supply of fish oil. Thus, there is great urgency to find and
implement sustainable alternatives to fish oil that can keep pace
with the growing global demand for fish products.
[0007] Many organizations recognize the limitations noted above
with respect to fish oil availability and aquaculture
sustainability. For example, in the United States, the National
Oceanic and Atmospheric Administration is partnering with the
Department of Agriculture in an Alternative Feeds Initiative to " .
. . identify alternative dietary ingredients that will reduce the
amount of fishmeal and fish oil contained in aquaculture fees while
maintaining the important human health benefits of farmed
seafood".
[0008] U.S. Pat. Appl. Pub. No. 2006-0115881-A1 suggests
recombinantly engineered Yarrowia lipolytica may be a useful
addition to most animal feeds, including aquaculture feeds, as a
means to provide necessary omega-3 and/or omega-6 PUFAs and based
on its unique protein:lipid:carbohydrate composition, as well as
unique complex carbohydrate profile (comprising an approximate
1:4:4.6 ratio of mannan:beta-glucans:chitin).
[0009] U.S. Pat. Appl. Pub. No. 2007/0226814 discloses fish food
containing at least one biomass obtained from fermenting
microorganisms wherein the biomass contains at least 20% DHA
relative to the total fatty acid content. Preferred microorganisms
used as sources for DHA are organisms belonging to the
Stramenopiles.
[0010] U.S. Pat. Appl. Pub. No. 2009/0202672 discloses, inter alia,
aquaculture feed incorporating oil obtained from a transgenic plant
engineered to produce stearidonic acid ["SDA"; 18:4 .omega.-3].
However, SDA is converted with low efficiency to DHA in fish.
[0011] If the growing aquaculture industry is to sustain its
contribution to world fish supplies, then it needs to reduce wild
fish inputs in feed and adopt more ecologically sound management
practices.
SUMMARY OF THE INVENTION
[0012] In one embodiment, the invention concerns an aquaculture
feed composition comprising:
[0013] (a) at least one source of eicosapentaenoic acid ["EPA"] and
optionally at least one source of docosahexanoic acid ["DHA"],
wherein said source can be the same or different; and,
[0014] (b) a ratio of concentration of EPA to concentration of DHA
which is greater than 2:1 based on the individual concentrations of
EPA and DHA in the aquaculture feed composition.
[0015] In a second embodiment, the invention concerns an
aquaculture feed composition wherein the aquaculture composition
further comprises a total amount of EPA and DHA that is at least
about 0.8% measured as a weight percent of the aquaculture feed
composition.
[0016] In a third embodiment, the invention concerns an aquaculture
feed composition wherein the at least one source of EPA is a first
source that is microbial oil and an optional second source that is
fish oil or fish meal.
[0017] In a fourth embodiment, the invention concerns an
aquaculture feed composition wherein the at least one source of DHA
is selected from the group consisting of microbial oil, fish oil,
fish meal, and a combination thereof.
[0018] In a fifth embodiment, the invention concerns an aquaculture
feed composition wherein the microbial oil is provided in a form
selected from the group consisting of biomass, processed biomass,
partially purified oil and purified oil, any of which is obtained
from at least one transgenic microbe engineered for the production
of polyunsaturated fatty acid-containing microbial oil comprising
EPA. Preferably, the at least one transgenic microbe is cultured.
The preferred transgenic microbe is Yarrowia lipolytica.
[0019] In a sixth embodiment, the invention concerns an aquaculture
feed composition wherein the biomass, processed biomass, partially
purified oil and/or purified oil are obtained from the cultured
transgenic microbe.
[0020] In a seventh embodiment, the invention concerns an
aquaculture feed composition which further comprises at least one
vegetable oil.
[0021] In an eighth embodiment, the invention concerns a method of
making an aquaculture feed composition comprising: [0022] a)
providing at least one source of EPA and optionally at least one
source of DHA, wherein said source can be the same or different;
[0023] b) providing additional feed components; and, [0024] c)
contacting (a) and (b) to make an aquaculture feed composition;
[0025] wherein said aquaculture feed composition has a ratio of
concentration of EPA to concentration of DHA which is greater than
2:1 based on the individual concentrations of EPA and DHA in the
aquaculture feed composition.
[0026] In a ninth embodiment, the invention concerns a method of
making an aquaculture feed composition wherein the aquaculture
composition further comprises a total amount of EPA and DHA that is
at least about 0.8% measured as weight percent of the aquaculture
feed composition.
DETAILED DESCRIPTION
[0027] All patents, patent applications, and publications cited
herein are incorporated by reference in their entirety.
[0028] In this disclosure, a number of terms and abbreviations are
used.
[0029] The following definitions are provided.
[0030] "Polyunsaturated fatty acid(s)" is abbreviated as
"PUFA(s)".
[0031] "Triacylglycerols" are abbreviated as "TAGs".
[0032] "Total fatty acids" are abbreviated as "TFAs".
[0033] "Fatty acid methyl esters" are abbreviated as "FAMEs".
[0034] "Dry cell weight" is abbreviated as "DCW".
[0035] As used herein the term "invention" or "present invention"
is intended to refer to all aspects and embodiments of the
invention as described in the claims and specification herein and
should not be read so as to be limited to any particular embodiment
or aspect.
[0036] The terms "aquaculture feed composition", "aquaculture feed
formulation", "aquaculture feed" and "aquafeed" are used
interchangeably herein. They refer to manufactured or artificial
diets (i.e., formulated feeds) to supplement or to replace natural
feeds in the aquaculture industry. These prepared foods are most
commonly produced in flake, pellet or tablet form. Typically, an
aquaculture feed composition refers to artificially compounded
feeds that are useful for farmed finfish and crustaceans (i.e.,
both lower-value staple food fish species [e.g., freshwater finfish
such as carp, tilapia and catfish] and higher-value cash crop
species for luxury or niche markets [e.g., mainly marine and
diadromous species such as shrimp, salmon, trout, yellowtail,
seabass, seabream and grouper]). These formulated feeds are
composed of several ingredients in various proportions
complementing each other to form a nutritionally complete diet for
the aquacultured species.
[0037] An aquaculture feed composition is used in the production of
an "aquaculture product", wherein the product is a harvestable
aquacultured species (e.g., finfish, crustaceans), which is often
sold for human consumption. For example, salmon are intensively
produced in aquaculture and thus are aquaculture products.
[0038] The term "aquaculture meat product" refers to food products
intended for human consumption comprising at least a portion of
meat from an aquaculture product as defined above. An aquaculture
meat product may be, for example, a whole fish or a filet cut from
a fish that is each sold as food. "Eicosapentaenoic acid" ["EPA"]
is the common name for cis-5,8,11,14,17-eicosapentaenoic acid. This
fatty acid is a 20:5 omega-3 fatty acid. The term EPA as used in
the present disclosure will refer to the acid or derivatives of the
acid (e.g., glycerides, esters, phospholipids, amides, lactones,
salts or the like) unless specifically mentioned otherwise.
[0039] "Docosahexaenoic acid" ["DHA"] is the common name for
cis-4,7,10,13,16,19-docosahexaenoic acid. This fatty acid is a 22:6
omega-3 fatty acid. The term DHA as used in the present disclosure
will refer to the acid or derivatives of the acid (e.g.,
glycerides, esters, phospholipids, amides, lactones, salts or the
like) unless specifically mentioned otherwise.
[0040] As used herein the term "biomass" refers to microbial
cellular material produced from the fermentation of a recombinant
production host producing EPA. Preferably, EPA is produced in
commercially significant amounts The preferred production host is a
recombinant strain of the oleaginous yeast, Yarrowia lipolytica.
The biomass may be in the form of whole cells, whole cell lysates,
homogenized cells, partially hydrolyzed cellular material, and/or
partially purified cellular material (e.g., microbially produced
oil). The term "processed biomass" refers to biomass that has been
subjected to additional processing such as drying, pasterization,
disruption, etc. All of which are discussed in greater detail
below.
[0041] The term "oleaginous" refers to those organisms that tend to
store their energy source in the form of lipid (Weete, In: Fungal
Lipid Biochemistry, 2.sup.nd Ed., Plenum, 1980). A class of plants
identified as oleaginous are commonly referred to as "oilseed"
plants. Examples of oilseed plants include, but are not limited to:
soybean (Glycine and Soja sp.), flax (Linum sp.), rapeseed
(Brassica sp.), maize, cotton, safflower (Carthamus sp.) and
sunflower (Helianthus sp.).
[0042] Within oleaginous microorganisms the cellular oil or TAG
content generally follows a sigmoid curve, wherein the
concentration of lipid increases until it reaches a maximum at the
late logarithmic or early stationary growth phase and then
gradually decreases during the late stationary and death phases
(Yongmanitchai and Ward, Appl. Environ. Microbiol. 57:419-25
(1991)).
[0043] The term "oleaginous yeast" refers to those microorganisms
classified as yeasts that make oil. It is not uncommon for
oleaginous microorganisms to accumulate in excess of about 25% of
their dry cell weight as oil. Examples of oleaginous yeast include,
but are no means limited to, the following genera: Yarrowia,
Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon
and Lipomyces.
[0044] The term "lipids" refer to any fat-soluble (i.e.,
lipophilic), naturally-occurring molecule. A general overview of
lipids is provided in U.S. Pat. Appl. Pub. No. 2009-0093543-A1 (see
Table 2 therein). The term "oil" refers to a lipid substance that
is liquid at 25.degree. C. and usually polyunsaturated. In
oleaginous organisms, oil constitutes a major part of the total
lipid. "Oil" is composed primarily of triacylglycerols ["TAGs"] but
may also contain other neutral lipids, phospholipids and free fatty
acids. The fatty acid composition in the oil and the fatty acid
composition of the total lipid are generally similar; thus, an
increase or decrease in the concentration of PUFAs in the total
lipid will correspond with an increase or decrease in the
concentration of PUFAs in the oil, and vice versa.
[0045] The term "extracted oil" refers to an oil that has been
separated from cellular materials, such as the microorganism in
which the oil was synthesized. Extracted oils are obtained through
a wide variety of methods, the simplest of which involves physical
means alone. For example, mechanical crushing using various press
configurations (e.g., screw, expeller, piston, bead beaters, etc.)
can separate oil from cellular materials. Alternatively, oil
extraction can occur via treatment with various organic solvents
(e.g., hexane), via enzymatic extraction, via osmotic shock, via
ultrasonic extraction, via supercritical fluid extraction (e.g.,
CO.sub.2 extraction), via saponification and via combinations of
these methods. An extracted oil does not require that it can not be
further purified or concentrated.
[0046] "Fish oil" refers to oil derived from the tissues of an oily
fish. Examples of oil fish include, but are not limited to:
menhaden, anchovy, cod and the like. Fish oil is a typical
component of feed used in aquaculture.
[0047] "Menhaden" refer to forage fish of the genera Brevoortia and
Ethmidium, two genera of marine fish in the family Clupeidae.
Recent taxonomic work using DNA comparisons have organized the
North American menhadens into large-scaled (Gulf and Atlantic
menhaden) and to small-scaled (Finescale and Yellowfin menhaden)
designations (Anderson, J. D., Fishery Bulletin,
105(3):368-378).
[0048] "Anchovies" from which anchovy fish meal and anchovy fish
oil are produced, are a family (Engraulidae) of small, common
salt-water forage fish. There are about 140 species in 16 genera,
found in the Atlantic, Indian, and Pacific Oceans.
[0049] "Vegetable oil" refers to any edible oil obtained from a
plant. Typically plant oil is extracted from seed or grain of a
plant.
[0050] The term "triacylglycerols" ["TAGs"] refers to neutral
lipids composed of three fatty acyl residues esterified to a
glycerol molecule. TAGs can contain long chain PUFAs and saturated
fatty acids, as well as shorter chain saturated and unsaturated
fatty acids.
[0051] "Neutral lipids" refer to those lipids commonly found in
cells in lipid bodies as storage fats and are so called because at
cellular pH, the lipids bear no charged groups. Generally, they are
completely non-polar with no affinity for water. Neutral lipids
generally refer to mono-, di-, and/or triesters of glycerol with
fatty acids, also called monoacylglycerol, diacylglycerol or
triacylglycerol, respectively, or collectively, acylglycerols. A
hydrolysis reaction must occur to release free fatty acids from
acylglycerols.
[0052] The term "total fatty acids" ["TFAs"] herein refer to the
sum of all cellular fatty acids that can be derivitized to fatty
acid methyl esters ["FAMEs"] by the base transesterification method
(as known in the art) in a given sample, which may be the biomass
or oil, for example. Thus, total fatty acids include fatty acids
from neutral lipid fractions (including diacylglycerols,
monoacylglycerols and TAGs) and from polar lipid fractions
(including, e.g., the phosphatidylcholine and
phosphatidylethanolamine fractions) but not free fatty acids.
[0053] The term "total lipid content" of cells is a measure of TFAs
as a percent of the dry cell weight ["DCW"], although total lipid
content can be approximated as a measure of FAMEs as a percent of
the DCW ["FAMEs % DCW"]. Thus, total lipid content ["TFAs % DCW"]
is equivalent to, e.g., milligrams of total fatty acids per 100
milligrams of DCW.
[0054] The concentration of a fatty acid in the total lipid is
expressed to herein as a weight percent of TFAs (% TFAs), e.g.,
milligrams of the given fatty acid per 100 milligrams of TFAs.
Unless otherwise specifically stated in the disclosure herein,
reference to the percent of a given fatty acid with respect to
total lipids is equivalent to concentration of the fatty acid as %
TFAs (e.g., % EPA of total lipids is equivalent to EPA % TFAs).
[0055] In some cases, it is useful to express the content of a
given fatty acid(s) in a cell as its weight percent of the dry cell
weight (% DCW). Thus, for example, eicosapentaenoic acid % DCW
would be determined according to the following formula:
(eicosapentaenoic acid % TFAs)*(TFAs % DCW)]/100. The content of a
given fatty acid(s) in a cell as its weight percent of the dry cell
weight (% DCW) can be approximated, however, as: (eicosapentaenoic
acid % TFAs)*(FAMEs % DCW)]/100.
[0056] The terms "lipid profile" and "lipid composition" are
interchangeable and refer to the amount of individual fatty acids
contained in a particular lipid fraction, such as in the total
lipid or the oil, wherein the amount is expressed as a weight
percent of TFAs. The sum of each individual fatty acid present in
the mixture should be 100.
[0057] The term "blended oil" refers to an oil that is obtained by
admixing, or blending, the extracted oil described herein with any
combination of, or individual, oil to obtain a desired composition.
Thus, for example, types of oils from different microbes can be
mixed together to obtain a desired PUFA composition. Alternatively,
or additionally, the PUFA-containing oils disclosed herein can be
blended with fish oil, vegetable oil or a mixture of both to obtain
a desired composition.
[0058] The term "fatty acids" refers to long chain aliphatic acids
(alkanoic acids) of varying chain lengths, from about C.sub.12 to
C.sub.22, although both longer and shorter chain-length acids are
known. The predominant chain lengths are between C.sub.16 and
C.sub.22. The structure of a fatty acid is represented by a simple
notation system of "X:Y", where X is the total number of carbon
["C"] atoms in the particular fatty acid and Y is the number of
double bonds. Additional details concerning the differentiation
between "saturated fatty acids" versus "unsaturated fatty acids",
"monounsaturated fatty acids" versus "polyunsaturated fatty acids"
["PUFAs"], and "omega-6 fatty acids" [".omega.-6" or "n-6"] versus
"omega-3 fatty acids" .omega.-3'' or "n-3"] are provided in U.S.
Pat. No. 7,238,482, which is hereby incorporated herein by
reference.
[0059] Nomenclature used to describe PUFAs herein is given in Table
1. In the column titled "Shorthand Notation", the omega-reference
system is used to indicate the number of carbons, the number of
double bonds and the position of the double bond closest to the
omega carbon, counting from the omega carbon, which is numbered 1
for this purpose. The remainder of the Table summarizes the common
names of .omega.-3 and .omega.-6 fatty acids and their precursors,
the abbreviations that will be used throughout the specification
and the chemical name of each compound.
TABLE-US-00001 TABLE 1 Nomenclature of Polyunsaturated Fatty Acids
And Precursors Shorthand Common Name Abbreviation Chemical Name
Notation Myristic -- tetradecanoic 14:0 Palmitic Palmitate
hexadecanoic 16:0 Palmitoleic -- 9-hexadecenoic 16:1 Stearic --
octadecanoic 18:0 Oleic -- cis-9-octadecenoic 18:1 Linoleic LA
cis-9,12-octadecadienoic 18:2 .omega.-6 .gamma.-Linolenic GLA
cis-6,9,12-octadecatrienoic 18:3 .omega.-6 Eicosadienoic EDA
cis-11,14-eicosadienoic 20:2 .omega.-6 Dihomo- .gamma.- DGLA
cis-8,11,14-eicosatrienoic 20:3 .omega.-6 Linolenic Arachidonic ARA
cis-5,8,11,14- 20:4 .omega.-6 eicosatetraenoic .alpha.-Linolenic
ALA cis-9,12,15- 18:3 .omega.-3 octadecatrienoic Stearidonic STA
cis-6,9,12,15- 18:4 .omega.-3 octadecatetraenoic Eicosatrienoic
ETrA cis-11,14,17- 20:3 .omega.-3 eicosatrienoic Eicosa- ETA
cis-8,11,14,17- 20:4 .omega.-3 tetraenoic eicosatetraenoic Eicosa-
EPA cis-5,8,11,14,17- 20:5 .omega.-3 pentaenoic eicosapentaenoic
Docosa- DTA cis-7,10,13,16- 22:4 .omega.-6 tetraenoic
docosatetraenoic Docosa- DPAn-6 cis-4,7,10,13,16- 22:5 .omega.-6
pentaenoic docosapentaenoic Docosa- DPA cis-7,10,13,16,19- 22:5
.omega.-3 pentaenoic docosapentaenoic Docosa- DHA
cis-4,7,10,13,16,19- 22:6 .omega.-3 hexaenoic docosahexaenoic
[0060] As used herein, "transgenic" or "genetically engineered"
refers to a microbe, plant or a cell which comprises within its
genome a heterologous polynucleotide. Preferably, the heterologous
polynucleotide is stably integrated within the genome such that the
polynucleotide is passed on to successive generations. The
heterologous polynucleotide may be integrated into the genome alone
or as part of an expression construct. Thus, transgenic is used
herein to include any microbe, cell, cell line, and/or tissue, the
genotype of which has been altered by the presence of heterologous
nucleic acid.
[0061] "Fish meal" refers to a protein source for aquaculture feed
compositions. Fish meals are typically either produced from fishery
wastes associated with the processing of fish for human consumption
(e.g., salmon, tuna) or produced from specific fish (i.e., herring,
menhaden, pollack) which are harvested solely for the purpose of
producing fish meal.
[0062] Aquaculture involves cultivating aquatic populations (e.g.,
freshwater and saltwater organisms) under controlled conditions.
Organisms grown in aquaculture may include fish and crustaceans.
Crustaceans are, for example, lobsters, crabs, shrimp, prawns and
crayfish. The farming of finfish is the most common form of
aquaculture. It involves raising fish commercially in tanks, ponds,
or ocean enclosures, usually for food. A facility that releases
juvenile fish into the wild for recreational fishing or to
supplement a species' natural numbers is generally referred to as a
fish hatchery. Particularly of interest are fish of the salmonid
group, for example, cherry salmon (Oncorhynchus masou), Chinook
salmon (Oncorhynchus tshawytscha), chum salmon (Oncorhynchus keta),
coho salmon (Oncorhynchus kisutch), pink salmon (Oncorhynchus
gorbuscha), sockeye salmon (Oncorhynchus nerka) and Atlantic salmon
(Salmo salar). Other finfish of interest for aquaculture include,
but are not limited to, various trout, as well as whitefish such as
tilapia (including various species of Oreochromis, Sarotherodon,
and Tilapia), grouper (subfamily Epinephelinae), sea bass, catfish
(order Siluriformes), bigeye tuna (Thunnus obesus), carp (family
Cyprimidae) and cod (genus Gadus).
[0063] Aquaculture typically requires a prepared aquaculture feed
composition to meet dietary requirements of the cultured animals.
Dietary requirements of different aquaculture species vary, as do
the dietary requirements of a single species during different
stages of growth. Thus, tremendous research is invested towards
optimizing each aquaculture feed composition for each stage of
growth of a cultured organism.
[0064] As an example, one can consider the 6-phase life cycle of
salmon. In the wild, the salmon life cycle begins with the
fertilization of spawned eggs. The eggs hatch into "alevin", which
live off the nutritious yolk sac that hangs off their undersides
for several months. Then, alevin develop into "fry", which feed
mainly on zooplankton until they grow large enough to eat aquatic
insects and other larger foods. When the fry are several months to
1 year old, they develop very noticeable markings along their
flanks. They are then termed salmon "parr", which feed mainly on
freshwater terrestrial and aquatic insects, amphipods, worms,
crustaceans, amphibian larvae, fish eggs, and young fish for 1 to 3
years. The process of smolting, which normally occurs when the fish
are 12-18 months old, enables the "smolts" to transition from a
freshwater environment to open salt water seas. Adult salmon feed
on smaller fish, such as herring, sandeels, pelagic amphipods and
krill while in the open ocean; they will return to the rivers in
which they were born after being at sea for 1-4 yr.
[0065] In aquaculture, salmon are typically farmed in two stages.
In the first stage, fish are hatched from eggs and raised in
freshwater tanks for 12-18 months to the smolt stage.
Alternatively, spawning channels, or artificial streams, may be
used in the first stage. In the second stage, the smolts are
transferred to floating sea cages or net pens which are anchored in
bays or fjords along a coast. Cages or pens are provided with feed
delivery equipment. Aquacultured animals may be fed different
aquaculture feed compositions that are formulated to meet the
changing nutrient requirements needed during different stages of
growth (Handbook of Salmon Farming; Stead and Laird (eds) (2002)
Praxis Publishing Ltd., Chichester, UK). The present aquaculture
feed compositions may be fed to animals to support their growth by
any method of aquaculture known by one skilled in the art ("Food
for Thought: the Use of Marine Resources in Fish Feed" Editor:
Tveferaas, head of conservation, WWF-Norway, Report #02/03
(2/2003)).
[0066] Once the aquaculture animals reach an appropriate size, the
crop is harvested, processed to meet consumer requirements, and can
be shipped to market, generally arriving within hours of leaving
the water.
[0067] For example, a common harvesting method for aquacultured
fish is to use a sweep net, which operates a bit like a purse seine
net. The sweep net is a big net with weights along the bottom edge.
It is stretched across the pen with the bottom edge extending to
the bottom of the pen. Lines attached to the bottom corners are
raised, herding some fish into the purse, where they are netted.
More advanced systems use a percussive-stun harvest system that
kills the fish instantly and humanely with a blow to the head from
a pneumatic piston. They are then bled by cutting the gill arches
and immediately immersed in iced water. Harvesting and killing
methods are designed to minimize scale loss, and avoid the fish
releasing stress hormones, which negatively affect flesh
quality.
[0068] To produce a salmon of harvestable size (i.e., 2.5-4 kg),
appropriate aquaculture feed compositions may be formulated as
appropriate over the dietary cycles of the salmon. Commercial feeds
generally rely on available supplies of fish oil to provide energy
and specific fatty acid requirements for aquacultured fish.
Generally, it takes between 3-7 kg, with the average around 5 kg,
of captured pelagic fish to provide the fish oil necessary to
produce one kg of salmon. Thus, the limited global supply of fish
oil will ultimately limit growth of aquaculture industries.
Additionally, removal of large numbers of smaller species of fish
from the food chain can have adverse ecosystem affects.
[0069] Aquaculture feed compositions are composed of micro and
macro components. In general, all components, which are used at
levels of more than 1%, are considered as macro components. Feed
ingredients used at levels of less than 1% are micro components.
They have to be premixed to achieve a homogeneous distribution of
the micro components in the complete feed. Both macro and micro
ingredients are subdivided into components with nutritional
functions and technical functions. Components with technical
functions improve the physical quality of the aquaculture feed
composition or its appearance.
[0070] Macro components with nutritional functions provide aquatic
animals with protein and energy required for growth and
performance. With respect to fish, the aquaculture feed composition
should ideally provide the fish with: 1) fats, which serve as a
source of fatty acids for energy (especially for heart and skeletal
muscles); and, 2) amino acids, which serve as building blocks of
proteins. Fats also assist in vitamin absorption; for example,
vitamins A, D, E and K are fat-soluble or can only be digested,
absorbed, and transported in conjunction with fats.
[0071] Carbohydrates, typically of plant origin (e.g., wheat,
sunflower meal, corn gluten, soybean meal), are also often included
in the feed compositions, although carbohydrates are not a superior
energy source for fish over protein or fat.
[0072] Fats are typically provided via incorporation of fish meals
(which contain a minor amount of fish oil) and fish oils into the
aquaculture feed compositions. Extracted oils that may be used in
aquaculture feed compositions include fish oils (e.g., from the
oily fish menhaden, anchovy, herring, capelin and cod liver), and
vegetable oil (e.g., from soybeans, rapeseeds, sunflower seeds and
flax seeds). Typically, fish oil is the preferred oil, because it
contains the long chain omega-3 polyunsaturated fatty acids
["PUFAs"], EPA and DHA; in contrast, vegetable oils do not provide
a source of EPA and/or DHA. These PUFAs are needed for growth and
health of most aquaculture products. A typical aquaculture feed
composition will comprise from about 15-30% of oil (e.g., fish,
vegetable, etc.), measured as a weight percent of the aquaculture
feed composition.
[0073] The amount of EPA (as a percent of total fatty acids ["%
TFAs"]) and DHA % TFAs provided in typical fish oils varies, as
does the ratio of EPA to DHA. Typical values are summarized in
Table 2, based on the work of Turchini, Torstensen and Ng (Reviews
in Aquaculture 1:10-57 (2009)):
TABLE-US-00002 TABLE 2 Typical EPA And DHA Content In Various Fish
Oils Fish Oil EPA DHA EPA:DHA Ratio Anchovy oil 17% 8.8% 1.93
Capelin oil 4.6% 3.0% 1.53 Menhaden oil 11% 9.1% 1.21 Herring oil
8.4% 4.9% 1.71 Cod liver oil 11.2% 12.6% 0.89
[0074] Often, oil from fish that are have lower EPA:DHA ratios is
used in aquaculture feed compositions, due to the lower cost.
Anchovy oil has the highest EPA:DHA ratio; however, using this oil
as the sole oil source in an aquaculture feed composition would
result in an EPA:DHA ratio of less than 2:1 in the final
formulation.
[0075] The protein supplied in aquaculture feed compositions can be
of plant or animal origin. For example, protein of animal origin
can be from marine animals (e.g., fish meal, fish oil, fish
protein, krill meal, mussel meal, shrimp peel, squid meal, squid
oil, etc.) or land animals (e.g., blood meal, egg powder, liver
meal, meat meal, meat and bone meal, silkworm, pupae meal, whey
powder, etc.). Protein of plant origin can include vegetable oil,
lecithin, rice and the like.
[0076] The technical functions of macro components are overlapping
as, for example, wheat gluten may be used as a pelleting aid and
for its protein content, which has a relatively high nutitional
value. There can also be mentioned guar gum and wheat flour.
[0077] Micro components include feed additives such as vitamins,
trace minerals, feed antibiotics and other biologicals. Minerals
used at levels of less than 100 mg/kg (100 ppm) are considered as
micro minerals or trace minerals.
[0078] Micro components with nutritional functions are all
biologicals and trace minerals. They are involved in biological
processes and are needed for good health and high performance.
There can be mentioned vitamins such as vitamins A, E, K.sub.3,
D.sub.3, B.sub.1, B.sub.3, B.sub.6, B.sub.12, C, biotin, folic
acid, panthothenic acid, nicotinic acid, choline chloride,
inositiol, para-amino-benzoic acid. There can be mentioned minerals
such as salts of calcium, cobalt, copper, iron, magnesium,
phosophorus, potasium, selenium and zinc. Other components may
include, but are not limited to, antioxidants, beta-glucans, bile
salt, cholesterol, enzymes, monosodium glutamate, etc.
[0079] The technical functions of micro ingredients are mainly
related to pelleting, detoxifying, mould prevention, antioxidation,
etc.
[0080] The present invention concerns a sustainable alternative to
fish oil. Specifically, the invention concerns an aquaculture feed
composition comprising: (a) at least one source of EPA and
optionally at least one source of DHA, wherein said source can be
the same or different; and, (b) a ratio of concentration of EPA to
concentration of DHA which is greater than 2:1 based on the
individual concentrations of EPA and DHA, each measured as a weight
percent of total fatty acids in the aquaculture feed
composition.
[0081] The aquaculture feed composition may further comprise a
total amount of EPA and DHA that is at least about 0.8%, measured
as weight percent of the aquaculture feed composition. This amount
(i.e., 0.8%) is typically an appropriate minimal concentration that
is suitable to support the growth of a variety of animals grown in
aquaculture, and particularly is suitable for inclusion in the
diets of salmonid fish.
[0082] As previously discussed, the highest EPA:DHA ratio in fish
oil (i.e., anchovy oil) was 1.93:1 (Turchini, Torstensen and Ng,
supra). Thus, it is believed that no commercially available
aquaculture feed composition has been produced having an EPA:DHA
ratio greater than 1.93:1. To achieve an EPA:DHA ratio greater than
2:1, as described herein, an alternate source of EPA (and
optionally DHA) is required. If no DHA is present in the
aquaculture feed composition, then the EPA:DHA ratio may be
considered to be greater than 2:1.
[0083] In preferred embodiments of the invention here, the
aquaculture feed composition comprises a microbial oil comprising
EPA. This may optionally be used in combination with fish oil or
fish meal (thereby effectively reducing the total amount of fish
oil or fish meal that is required in the feed formulation, while
maintaining desired EPA content). The microbial oil comprising EPA
may also contain DHA; or, DHA may be obtained from a second
microbial oil, fish oil, fish meal, and combinations thereof. In
some formulations, the microbial oil comprising EPA may be
supplemented with a vegetable oil, to reach the desired total
oil/fat content.
[0084] EPA can be produced microbially via numerous different
processes, based on the natural abilities of the specific microbial
organism utilized [e.g., heterotrophic diatoms Cyclotella sp. and
Nitzschia sp. (U.S. Pat. No. 5,244,921); Pseudomonas, Alteromonas
or Shewanella species (U.S. Pat. No. 5,246,841); filamentous fungi
of the genus Pythium (U.S. Pat. No. 5,246,842); or Mortierella
elongata, M. exigua, or M. hygrophila (U.S. Pat. No. 5,401,646)].
One of skill in the art will be able to identity other microbes
which have the native ability to produce EPA, based on phenotypic
analysis, GC analysis of the PUFA products, review of available
public and patent literature and screening of microbes related to
those previously identified as EPA-producers. Microbial oils
comprising EPA from these organisms may be provided in a variety of
forms for use in the aquaculture feed compositions herein, wherein
the oil is typically contained within microbial biomass or
processed biomass, or the oil is partially purified or purified
oil. In most cases, it will be most cost effective to incorporate
microbial biomass or processed biomass into the aquaculture feed
composition, as opposed to the microbial oil (in partial or
purified form); however, these economics should not be considered
as a limitation herein.
[0085] Alternately, microbial oil comprising EPA can be produced in
transgenic microbes engineered for the production of
polyunsaturated fatty acid-containing microbial oil comprising EPA.
Microbes such as algae, fungi, yeast, stramenopiles and bacteria
may be engineered for production of PUFAs, including EPA, by
integration of appropriate heterologous genes encoding desaturases
and elongases of either the delta-6 desaturase/delta-6 elongase
pathway or the delta-9 elongase/delta-8 desaturase pathway into the
host organism. The particular genes included within a particular
expression cassette depend on the host organism, its PUFA profile
and/or desaturase/elongase profile, the availability of substrate
and the desired end product(s). A PUFA polyketide synthase ["PKS"]
system that produces EPA, such as that found in e.g., Shewanella
putrefaciens (U.S. Pat. No. 6,140,486), Shewanella olleyana (U.S.
Pat. No. 7,217,856), Shewanella japonica (U.S. Pat. No. 7,217,856)
and Vibrio marinus (U.S. Pat. No. 6,140,486), could also be
introduced into a suitable microbe to enable EPA, and optionally
DHA, production. Other PKS systems that natively produce DHA could
also be engineered to enable is only EPA or a suitable combination
of the PUFAs to yield an EPA:DHA ratio of greater than 2:1.
[0086] One skilled in the art is familiar with the considerations
and techniques necessary to introduce one or more expression
cassettes encoding appropriate enzymes for EPA biosynthesis into a
microbial host organism of choice, and numerous teachings are
provided in the literature to one of skill. Microbial oils
comprising EPA from these genetically engineered organisms may also
be suitable for use in the aquaculture feed compositions herein,
wherein the oil may be contained within the microbial biomass or
processed biomass, or the oil may be partially purified or purified
oil.
[0087] In some applications, the microbe engineered for EPA
production is is oleaginous, i.e., the organism tends to store its
energy source in the form of lipid (Weete, In: Fungal Lipid
Biochemistry, 2.sup.nd Ed., Plenum, 1980). Oleaginous yeast are a
preferred microbe, as these microorganisms can commonly accumulate
in excess of about 25% of their dry cell weight as oil. Examples of
oleaginous yeast include, but are by no means limited to, the
following genera: Yarrowia, Candida, Rhodotorula, Rhodosporidium,
Cryptococcus, Trichosporon and Lipomyces. More specifically,
illustrative oil-synthesizing yeasts include: Rhodosporidium
toruloides, Lipomyces starkeyii, L. lipoferus, Candida revkaufi, C.
puicherrima, C. tropicalis, C. utilis, Trichosporon pullans, T.
cutaneum, Rhodotorula glutinus, R. graminis, and Yarrowia
lipolytica (formerly classified as Candida lipolytica). Most
preferred is the oleaginous yeast Yarrowia lipolytica. Examples of
suitable Y. lipolytica strains include, but are not limited to, Y.
lipolytica strains designated as ATCC #20362, ATCC #8862, ATCC
#18944, ATCC #76982 and/or LGAM S(7)1 (Papanikolaou S., and Aggelis
G., Bioresour. Technol. 82(1):43-9 (2002)).
[0088] Some references describing means to engineer the oleaginous
host organism Yarrowia lipolytica for EPA biosynthesis are provided
as follows: U.S. Pat. No. 7,238,482, U.S. Pat. No. 7,550,286, U.S.
Pat. Appl. Pub. No. 2006-0115881-A1, U.S. Pat. Appl. Pub. No.
2009-0093543-A1, U.S. patent application Ser. No. 12/814,815 (E.I.
duPont de Nemours & Co., Inc., Attorney Docket No.
"CL4674USNA", filed Jun. 14, 2010 and hereby incorporated herein by
reference) and U.S. patent application Ser. No. 12/814,880 (E.I. du
Pont de Nemours & Co., Inc., Attorney Docket Number
"CL4714USNA", filed Jun. 14, 2010 and hereby incorporated herein by
reference). This list is not exhaustive and should not be construed
as limiting.
[0089] It may be desirable for the oleaginous yeast to be capable
of "high-level EPA production", wherein the organism can produce at
least about 5-10% of EPA in the total lipids. More preferably, the
oleaginous yeast will produce at least about 10-25% of EPA in the
total lipids, more preferably at least about 25-35% of EPA in the
total lipids, more preferably at least about 35-45% of EPA in the
total lipids, more preferably at least about 45-55% of EPA in the
total lipids, and most preferably at least about 55-60% of EPA in
the total lipids. The structural form of the EPA is, not limiting;
thus, for example, EPA may exist in the total lipids as free fatty
acids or in esterified forms such as acylglycerols, phospholipids,
sulfolipids or glycolipids.
[0090] For example, U.S. Pat. Appl. Pub. No. 2009-0093543-A1
describes high-level EPA production in optimized recombinant
Yarrowia lipolytica strains. Specifically, strains are disclosed
having the ability to produce microbial oils comprising at least
about 43.3 EPA % TFAs, with less than about 23.6 LA % TFAs (an
EPA:LA ratio of 1.83) and less than about 9.4 oleic acid (18:1)
TFAs. The preferred strain was Y4305, whose maximum production was
55.6 EPA % TFAs, with an EPA:LA ratio of 3.03. Generally, the
EPA-producing strains of U.S. Pat. Appl. Pub. No. 2009-0093543-A1
comprised the following genes of the omega-3/omega-6 fatty acid
biosynthetic pathway: a) at least one gene encoding delta-9
elongase; b) at least one gene encoding delta-8 desaturase; c) at
least one gene encoding delta-5 desaturase; d) at least one gene
encoding delta-17 desaturase; e) at least one gene encoding
delta-12 desaturase; f) at least one gene encoding C.sub.16/18
elongase; and, g) optionally, at least one gene encoding
diacylglycerol cholinephosphotransferase ["CPT1"]. Since the
pathway is genetically engineered into the host cell, there is no
DHA concomitantly produced due to the lack of the appropriate
enzymatic activities for elongation of EPA to DPA (catalyzed by a
C.sub.20/22 elongase) and desaturation of DPA to DHA (catalyzed by
a delta-4 desaturase). The disclosure also described microbial oils
obtained from these engineered yeast strains and oil concentrates
thereof.
[0091] A derivative of Yarrowia lipolytica strain Y4305 is
described herein, known as Y. lipolytica strain Y4305 F1B1. Upon
growth in a two liter fermentation (parameters similar to those of
U.S. Pat. Appl. Pub. No. 2009-009354-A1, Example 10), average EPA
productivity ["EPA % DCW"] for strain Y4305 was 50-56, as compared
to 50-52 for strain Y4305-F1B1. Average lipid content ["TFAs %
DCW"] for strain Y4305 was 20-25, as compared to 28-32 for strain
Y4305-F1B1. Thus, lipid content was increased 29-38% in strain
Y4503-F1B1, with minimal impact upon EPA productivity.
[0092] More recently, U.S. patent application Ser. No. 12/814,815
(E.I. duPont de Nemours & Co., Inc., Attorney Docket No.
"CL4674USNA", filed Jun. 14, 2010) and U.S. patent application Ser.
No. 12/814,880 (E.I. du Pont de Nemours & Co., Inc., Attorney
Docket Number "CL4714USNA", filed Jun. 14, 2010) teach optimized
strains of recombinant Yarrowia lipolytica having the ability to
produce further improved microbial oils relative to those strains
described in U.S. Pat. Appl. Pub. No. 2009-0093543-A1, based on the
EPA % TFAs and the ratio of EPA:LA. In addition to expressing genes
of the omega-3/omega-6 fatty acid biosynthetic pathway as detailed
in U.S. Pat. Appl. Pub. No. 2009-0093543-A1, these improved strains
are distinguished by: a) comprising at least one multizyme, wherein
said multizyme comprises a polypeptide having at least one fatty
acid delta-9 elongase linked to at least one fatty acid delta-8
desaturase [a "DGLA synthase"]; b) optionally comprising at least
one polynucleotide encoding an enzyme selected from the group
consisting of a malonyl CoA synthetase or an acyl-CoA
lysophospholipid acyltransferase ["LPLAT"]; c) comprising at least
one peroxisome biogenesis factor protein whose expression has been
down-regulated; d) producing at least about 50 EPA % TFAs; and, e)
having a ratio of EPA:LA of at least about 3.1.
[0093] Specifically, in addition to possessing at least about 50
EPA % TFAs, the lipid profile within the improved optimized strains
of Yarrrowia lipolytica of U.S. patent application Ser. No.
12/814,815 and U.S. patent application Ser. No. 12/814,880, or
within extracted or unconcentrated oil therefrom, will have a ratio
of EPA % TFAs to LA % TFAs of at least about 3.1. Lipids produced
by the improved optimized recombinant Y. lipolytica strains are
also distinguished as having less than 0.5% GLA or DHA (when
measured by GC analysis using equipment having a detectable level
down to about 0.1%) and having a saturated fatty acid content of
less than about 8%. This low percent of saturated fatty acids
(i.e., 16:0 and 18:0) benefits both humans and animals.
[0094] Thus, it is considered that the EPA oils described above
from genetically engineered strains of Yarrowia lipolytica are
substantially free of DHA, low in saturated fatty acids and high in
EPA. Example 6 herein provides a summary of some representative
strains of Yarrowia lipolytica engineered to produce high levels of
EPA. Furthermore, the cited art provides numerous examples of
additional suitable microbial strains and species, comprising EPA
and having an EPA:DHA ratio of greater than 2:1. It is also
contemplated herein that any of these microbes could be subjected
to further genetic engineering improvements and thus be a suitable
source of EPA in the aquaculture feed compositions and methods
described herein.
[0095] The aquaculture feed compositions of the present invention
optionally comprise at least one source of DHA (i.e., in addition
to the at least one source of EPA discussed supra). The source of
DHA can be the same or different than that of EPA, although the
ratio of EPA:DHA must be greater than 2:1 based on the individual
concentrations of EPA and DHA, each measured as a weight percent of
total fatty acids in the aquaculture feed composition.
[0096] In preferred embodiments, at least one source of DHA is
selected from the group consisting of: microbial oil, fish oil,
fish meal, and combinations thereof.
[0097] Fish oil is typically a source of DHA, as well as of EPA, in
aquaculture feed compositions (Table 2, supra). Fish meal is also
often incorporated into aquaculture feed compositions as a protein
source. Since this is a fish product, the meals have a low oil
content and thereby can provide a small portion of PUFAs to the
total aquaculture feed composition, in addition to that provided
directly as fish oil.
[0098] DHA can be produced using processes based on the natural
abilities of native microbes. See, e.g., processes developed for
Schizochytrium species (U.S. Pat. No. 5,340,742; U.S. Pat. No.
6,582,941); Ulkenia (U.S. Pat. No. 6,509,178); Pseudomonas sp.
YS-180 (U.S. Pat. No. 6,207,441); Thraustochytrium genus strain
LFF1 (U.S. 2004/0161831 A1); Crypthecodinium cohnii (U.S. Pat.
Appl. Pub. No. 2004/0072330 A1; de Swaaf, M. E. et al. Biotechnol
Bioeng., 81(6):666-72 (2003) and Appl Microbiol Biotechnol.,
61(1):40-3 (2003)); Emiliania sp. (Japanese Patent Publication
(Kokai) No. 5-308978 (1993)); and Japonochytrium sp. (ATCC #28207;
Japanese Patent Publication (Kokai) No. 199588/1989)].
Additionally, the following microorganisms are known to have the
ability to produce DHA: Vibrio marinus (a bacterium isolated from
the deep sea; ATCC #15381); the micro-algae Cyclotella cryptica and
Isochrysis galbana; and, flagellate fungi such as Thraustochytrium
aureum (ATCC #34304; Kendrick, Lipids, 27:15 (1992)) and the
Thraustochytrium sp. designated as ATCC #28211, ATCC #20890 and
ATCC #20891. Currently, there are at least three different
fermentation processes for commercial production of DHA:
fermentation of C. cohnii for production of DHASCO.TM. (Martek
Biosciences Corporation, Columbia, Md.); fermentation of
Schizochytrium sp. for production of an oil formerly known as
DHAGoId (Martek Biosciences Corporation); and fermentation of
Ulkenia sp. for production of DHActive.TM. (Nutrinova, Frankfurt,
Germany). As such, microbial oils comprising DHA from any of these
organisms may be provided in a variety of forms for use in the
aquaculture feed compositions herein, wherein the oil is typically
contained within microbial biomass or processed biomass, or the oil
is partially purified or purified oil.
[0099] Similarly, means to genetically engineer a microbe such that
it is capable of DHA production will be well known to one of skill
in the art. Only two additional enzymatic steps are required to
convert EPA to DHA and thus integration of appropriate heterologous
genes encoding C.sub.20/22 elongase and delta-4 desaturase will be
readily possible, using the teachings described above for
engineering EPA.
[0100] Of particular import, the microbial oil may comprise a
mixture of EPA and DHA to achieve the most desired ratio of EPA:DHA
in the final aquaculture feed composition. Based on an increasing
emphasis on the ability to engineer microorganisms for production
of "designer" lipids and oils, wherein the fatty acid content and
composition are carefully specified by genetic engineering for a
variety of purposes, it is contemplated that a suitable microbe
could be engineered producing a combination of EPA and DHA. For
example, one is referred to U.S. Pat. No. 7,550,286, wherein
recombinant Yarrowia lipolytica strains are disclosed having the
ability to produce microbial oils comprising at least about 4.7 EPA
% TFAs, 18.3 DPA % TFAs and 5.6 DHA % TFAs. Although this
particular example fails to provide a microbial oil having an
EPA:DHA ratio of greater than 2:1, subsequent genetic engineering
could readily modify the overall lipid profile. Or, this microbial
oil could be mixed with microbial oil from an alternate Yarrowia
lipolytica strain producing high EPA to achieve the preferred
target ratio. One of skill in the art will readily appreciate the
numerous alternatives that are disclosed herein, as a means to
obtain a microbial oil comprising at least one source of EPA and
optionally at least one source of DHA, wherein the EPA:DHA ratio is
greater than 2:1.
[0101] When a microbe (or combination of microbes) are used in the
present invention as a source of EPA and/or DHA, the microbe will
be grown under standard conditions well known by one skilled in the
art of microbiology or fermentation science to optimize the
production of the PUFA. With respect to genetically engineered
microbes, the microbe will be grown under conditions that optimize
expression of chimeric genes (e.g., encoding desaturases,
elongases, acyltransferases, etc.) and produce the greatest and the
most economical yield of EPA and/or DHA. Thus, a genetically
engineered microbe producing lipids containing the desired PUFA may
be cultured and grown in a fermentation medium under conditions
whereby the PUFA is produced by the microorganism. Typically, the
microorganism is fed with a carbon and nitrogen source, along with
a number of additional chemicals or substances that allow growth of
the microorganism and/or production of the PUFA. The fermentation
conditions will depend on the microorganism used and may be
optimized for a high content of the PUFA in the resulting
biomass.
[0102] In general, media conditions may be optimized by modifying
the type and amount of carbon source, the type and amount of
nitrogen source, the carbon-to-nitrogen ratio, the amount of
different mineral ions, the oxygen level, growth temperature, pH,
length of the biomass production phase, length of the oil
accumulation phase and the time and method of cell harvest.
[0103] More specifically, fermentation media should contain a
suitable carbon source, such as are taught in U.S. Pat. No.
7,238,482 and U.S. Pat. Pub. No. 2009-0325265-A1. Although it is
contemplated that the source of carbon utilized for growth of an
engineered EPA-producing microbe may encompass a wide variety of
carbon-containing sources, preferred carbon sources are sugars,
glycerol and/or fatty acids. Most preferred are glucose, sucrose,
invert sucrose, fructose and/or fatty acids containing between
10-22 carbons. For example, the fermentable carbon source can be
selected from the group consisting of invert sucrose (i.e., a
mixture comprising equal parts of fructose and glucose resulting
from the hydrolysis of sucrose), glucose, fructose and combinations
of these, provided that glucose is used in combination with invert
sucrose and/or fructose.
[0104] Nitrogen may be supplied from an inorganic (e.g.,
(NH.sub.4).sub.2SO.sub.4) or organic (e.g., urea or glutamate)
source. In addition to appropriate carbon and nitrogen sources, the
fermentation media must also contain suitable minerals, salts,
cofactors, buffers, vitamins and other components known to those
skilled in the art suitable for the growth of the EPA-producing
microbe and promotion of the enzymatic pathways necessary for EPA
production. Particular attention is given to several metal ions
(e.g., Fe.sup.+2, Cu.sup.+2, Mn.sup.+2, Co.sup.+2, Zn.sup.+2 and
Mg.sup.+2) that promote synthesis of lipids and PUFAs (Nakahara, T.
et al., Ind. Appl. Single Cell Oils, D. J. Kyle and R. Colin, eds.
pp 61-97 (1992)).
[0105] Preferred growth media are common commercially prepared
media, such as Yeast Nitrogen Base (DIFCO Laboratories, Detroit,
Mich.). Other defined or synthetic growth media may also be used
and the appropriate medium for growth of Yarrowia lipolytica will
be known by one skilled in the art of microbiology or fermentation
science. A suitable pH range for the fermentation is typically
between about pH 4.0 to pH 8.0, wherein pH 5.5 to pH 7.5 is
preferred as the range for the initial growth conditions. The
fermentation may be conducted under aerobic or anaerobic
conditions.
[0106] Typically, accumulation of high levels of PUFAs in
oleaginous yeast cells requires a two-stage process, since the
metabolic state must be "balanced" between growth and
synthesis/storage of fats. Thus, most preferably, a two-stage
fermentation process is necessary for the production of EPA in
Yarrowia lipolytica. This approach is described in U.S. Pat. No.
7,238,482, as are various suitable fermentation process designs
(i.e., batch, fed-batch and continuous) and considerations during
growth.
[0107] When the desired amount of EPA and/or DHA has been produced
by the microorganism, the fermentation medium may be treated to
obtain microbial biomass comprising the PUFA. For example, the
fermentation medium may be filtered or otherwise treated to remove
at least part of the aqueous component. The fermentation medium
and/or the microbial biomass may be further processed, for example
the microbial biomass may be pasteurized or treated via other means
to reduce the activity of endogenous microbial enzymes that can
harm the microbial oil and/or PUFA products. The microbial biomass
may be subjected to drying (e.g., to a desired water content) or a
means of mechanical disruption (e.g., via physical means such as
bead beaters, screw extrusion, etc. to provide greater
accessibility to the cell contents), or a combination of these. The
microbial biomass may be granulated or pelletized for ease of
handling. A brief review of downstream processing is also available
by A. Singh and O. Ward (Adv. Appl. Microbiol., 45:271-312
(1997)).
[0108] Thus, micobial biomass obtained from any of the means
described above may be used as a source of microbial oil comprising
EPA and/or DHA for use in the aquaculture feed compositions
described herein.
[0109] In some embodiments, the PUFAs may be extracted from the
host cell through a variety of means well-known in the art. This
may be useful, since PUFAs, including EPA, may be found in the host
microorganism as free fatty acids or in esterified forms such as
acylglycerols, phospholipids, sulfolipids or glycolipids. One
review of extraction techniques, quality analysis and acceptability
standards for yeast lipids is that of Z. Jacobs (Critical Reviews
in Biotechnology, 12(5/6):463-491 (1992)). In general, extraction
may be performed with organic solvents, sonication, supercritical
fluid extraction (e.g., using carbon dioxide), saponification and
physical means such as presses, or combinations thereof. One is
referred to the teachings of U.S. Pat. No. 7,238,482 for additional
details.
[0110] Thus, microbial oil, whether partially purified or purfied,
obtained from any of the means described above may be used as a
source of EPA and/or DHA for use in the aquaculture feed
compositions described herein. Preferably, the microbial oil will
be used as a replacement of at least a portion of the fish oil that
would be used in a similar aquaculture feed composition.
[0111] The present invention also concerns a method of making an
aquaculture feed composition comprising: [0112] a) providing at
least one source of EPA and, optionally, at least one source of
DHA, wherein said source can be the same or different; [0113] b)
providing additional feed components; and, [0114] c) contacting (a)
and (b) to make an aquaculture feed composition;
[0115] wherein said aquaculture feed composition has a ratio of
concentration of EPA to concentration of DHA which is greater than
2:1 based on the individual concentrations of EPA and DHA in the
aquaculture feed composition.
[0116] In preferred embodiments, the at least one source of EPA is
a first source that is microbial oil and an optional second source
that is fish oil or fish meal. The at least one source of DHA is
selected from the group consisting of: microbial oil, fish oil,
fish meal, and combinations thereof.
[0117] One of skill in the art will be able to determine the
appropriate amount of microbial oil comprising EPA and optionally
DHA to be included in an aquaculture feed composition, to increase
the EPA:DHA ratio of the resulting aquaculture feed composition to
greater than 2:1 and, preferably, to result in a total amount of
EPA and DHA that is at least about 0.8%, measured as a weight
percent of the aquaculture feed composition. The microbial oil may
be included in an aquaculture feed as partially purified or
purified oil, or the microbial oil may be contained within
microbial biomass or processed biomass that is included.
[0118] The amount of microbial oil, or biomass containing microbial
oil, needed to achieve an EPA:DHA ratio of greater than 2:1 will
vary depending on factors. Determinants include consideration of
the EPA % TFAs, the EPA % DCW, the DHA % TFAs and the DHA % DCW of
the microbial biomass comprising the oil, the EPA % TFAs and DHA %
TFAs of a purified or partially purified oil, the content of EPA
and DHA in other components to be added to the aquaculture feed
composition (e.g., fishmeal, fish oil, vegetable oil, microalgae
oil), etc.
[0119] Exemplary calculations of EPA content, DHA content and
EPA:DHA ratios in aquaculture feed compositions are provided in
Example 4 (infra), based on formulation with variable
concentrations (i.e., 10%, 20% And 30%) of Yarrowia lipolytica
Y4305 F1B1 biomass, which was assumed to contain 15 EPA % DCW, 50
EPA % TFAs and 0.0 DHA % TFAs. More specifically, various
calculations are provided to demonstrate how this microbial biomass
containing EPA could readily be mixed with variable concentrations
of either anchovy oil or menhaden oil (0%, 2%, 5%, 10% and 20%), to
result in aquaculture feed compositions comprising from 1.8% to
10.02% total EPA and DHA in the final composition, with EPA:DHA
ratios ranging from 1.94:1 up to 47.7:1.
[0120] For example, if an aquaculture feed composition is prepared
comprising anchovy fishmeal (25% of total weight), anchovy oil (20%
of total weight) and Yarrowia lipolytica Y4305 F1B1 biomass that
provides 15 EPA % DCW (10% of total weight), the EPA:DHA ratio is
calculated to be 2.69:1. With less anchovy oil and/or more Y.
lipolytica Y4305 F1B1 biomass, the EPA:DHA ratio increases. In
another example, if an aquaculture feed composition is prepared
comprising menhaden fishmeal (25% of total weight), menhaden oil
(10% of total weight) and with Yarrowia lipolytica Y4305 F1B1
biomass that provides 15 EPA % DCW (10% of total weight), EPA:DHA
ratio is calculated to be 2.61:1. If fish oil is not used in the
aquaculture feed composition, as seen in the scenarios using no
anchovy oil or menhaden oil, then DHA will be available in the
final composition only as a result of fishmeal; this leads to even
higher EPA:DHA ratios.
[0121] Thus, Example 4 clearly demonstrates that a variety of
aquaculture feed compositions can be formulated, using different
amounts of various fish oils, in combination with different amounts
of microbial biomass containing EPA, to result in a range of
EPA:DHA ratios in the final aquaculture feed composition that are
greater than 2:1. Similar calculations may be made for microbial
biomass samples that contain various percents of EPA and/or in
alternate feed formulations that comprise vegetable oils, etc. In
this manner, various aquaculture feed compositions may be designed,
by one skilled in the art, that have an EPA:DHA ratio of greater
than 2:1. EPA:DHA ratios in the present aquaculture feed
composition are greater than 2:1, and may be at least about 2.2:1,
2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1,
8:1, 8.5:1, 9:1, 9.5:1, or 10:1 or higher. Although preferred
EPA:DHA ratios are described above, useful examples of EPA:DHA
ratios include any integer or portion thereof that is greater than
2:1.
[0122] Based on the disclosure herein, it will be clear that
renewable alternatives to fish oil can be utilized as a means to
produce aquaculture feed compositions. These modified formulations
do not impact fish health and may yield economic benefits to those
performing aquaculture. Additionally, the modified formulations of
the present invention will have societal benefits, as they will
support sustainable aquaculture. Implementing sustainable
alternatives to fish oil that can keep pace with the growing global
demand for aquaculture products will also be advantageous.
EXAMPLES
[0123] The present invention is further defined in the following
Examples. It should be understood that these Examples, while
indicating preferred embodiments of the invention, are given by way
of illustration only. It will be understood by those skilled in the
art that the invention is capable of numerous modifications,
substitutions, and rearrangements without departing from the spirit
of essential attributes of the invention. Reference should be made
to the appended claims, rather than to the foregoing specification,
as indicating the scope of the invention.
[0124] All aquaculture feed formulations and feed ingredients were
obtained from and/or produced by Nofima Ingrediens, Kierreidviken
16, NO-5141 Fvllingsdalen, Norway ("Nofima"). Thus, fish meal;
sunflower meal; hydrolyzed feather meal; corn gluten; soybean meal;
wheat; Carophyll Pink comprising 10% astaxanthin; and yttrium oxide
were obtained from Nofima.
[0125] The meaning of abbreviations is as follows: "kb" means
kilobase(s), "bp" means base pairs, "nt" means nucleotide(s), "hr"
means hour(s), "min" means minute(s), "sec" means second(s), "d"
means day(s), "L" means liter(s), "ml" means milliliter(s), ".mu.L"
means microliter(s), ".mu.g" means microgram(s), "ng" means
nanogram(s), "mM" means millimolar, ".mu.M" means micromolar, "nm"
means nanometer(s), ".mu.mol" means micromole(s), "DCW" means dry
cell weight, "TFAs" means total fatty acids and "FAMEs" means fatty
acid methyl esters.
General Methods
[0126] Lipid Analysis: Lipids were extracted using the Folch method
(Folch et al., J. Biol. Chem., 226:497 (1957)). Following
extraction, the chloroform phase was dried under N.sub.2 and the
residual lipid extract was redissolved in benzene, and then
transmethylated overnight with 2,2-dimethoxypropane and methanolic
HCl at room temperature, as described by Mason, M. E. and G. R.
Waller (J. Agric. Food Chem., 12:274-278 (1964)) and by Hoshi et
al. (J. Lipid Res., 14:599-601 (1973)). The methyl esters of fatty
acids thus formed were separated in a gas chromatograph (Hewlett
Packard 6890) with a split injector, a SGE BPX70 capillary column
(having a length of 60 m, an internal diameter of 0.25 mm and a
film thickness of 0.25 m) with flame ionization detector. The
carrier gas was helium. The injector and detector temperatures were
280.degree. C. The oven temperature was raised from 50.degree. C.
to 180.degree. C. at the rate of 10.degree. C./min, and then raised
to 240.degree. C. at the rate of 0.7.degree. C./min. All GC results
were analyzed using HP ChemStation software (Hewlett-Packard Co.).
The relative quantity of each fatty acid present was determined by
measuring the area under the peak of the FAME corresponding to that
fatty acid, and calculating the percentage relative to the sum of
all integrated peaks.
[0127] Yarrowia lipolytica Strains: Y. lipolytica strain Y4305 was
derived from wild type Yarrowia lipolytica ATCC #20362. Strain
Y4305 was previously described in U.S. Pat. Appl. Pub. No.
2009-0093543-A1, the disclosure of which is hereby incorporated in
its entirety. The final genotype of strain Y4305 with respect to
wild type Yarrowia lipolytica ATCC #20362 is SCP2-(YALI0E01298g),
YALI0C18711g-, Pex10-, YALI0F24167g-, unknown 1-, unknown 3-,
unknown 8-, GPD::FmD12::Pex20, YAT1::FmD12::OCT,
GPM/FBAIN::FmD12S::OCT, EXP1::FmD12S::Aco, YAT1::FmD12S::Lip2,
YAT1::ME3S::Pex16, EXP1::ME3S::Pex20 (3 copies), GPAT::EgD9e::Lip2,
EXP1::EgD9eS::Lip1, FBAINm::EgD9eS::Lip2, FBA::EgD9eS::Pex20,
GPD::EgD9eS::Lip2, YAT1::EgD9eS::Lip2, YAT1::E389D9eS::OCT,
FBAINm::EgD8M::Pex20, FBAIN::EgD8M::Lip1 (2 copies),
EXP1::EgD8M::Pex16, GPDIN::EgD8M::Lip1, YAT1::EgD8M::Aco,
FBAIN::EgD5::Aco, EXP1::EgD5S::Pex20, YAT1::EgD5S::Aco,
EXP1::EgD5S::ACO, YAT1::RD5S::OCT, YAT1::PaD17S::Lip1,
EXP1::PaD17::Pex16, FBAINm::PaD17::Aco, YAT1::YICPT1::ACO,
GPD::YICPT1::ACO. Chimeric genes in the above strain genotype are
represented by the notation system "X::Y::Z", where X is the
promoter region, Y is the coding region, and Z is the terminator,
which are all operably linked to one another. Abbreviations are as
follows: FmD12 is a Fusarium moniliforme delta-12 desaturase coding
region [U.S. Pat. No. 7,504,259]; FmD12S is a codon-optimized
delta-12 desaturase coding region derived from Fusarium moniliforme
(U.S. Pat. No. 7,504,259); ME3S is a codon-optimized C.sub.16/18
elongase coding region derived from Mortierella alpina (U.S. Pat.
No. 7,470,532); EgD9e is a Euglena gracilis delta-9 elongase coding
region (U.S. Pat. No. 7,645,604); EgD9eS is a codon-optimized
delta-9 elongase coding region derived from Euglena gracilis (U.S.
Pat. No. 7,645,604); E389D9eS is a codon-optimized delta-9 elongase
coding region derived from Eutreptiella sp. CCMP389 (U.S. Pat. No.
7,645,604); EgD8M is a synthetic mutant delta-8 desaturase coding
region (U.S. Pat. No. 7,709,239) derived from Euglena gracilis
(U.S. Pat. No. 7,256,033); EgD5 is a Euglena gracilis delta-5
desaturase coding region (U.S. Pat. No. 7,678,560); EgD5S is a
codon-optimized delta-5 desaturase coding region derived from
Euglena gracilis (U.S. Pat. No. 7,678,560); RD5S is a
codon-optimized delta-5 desaturase coding region derived from
Peridinium sp. CCMP626 (U.S. Pat. No. 7,695,950), PaD17 is a
Pythium aphanidermatum delta-17 desaturase coding region (U.S. Pat.
No. 7,556,949); PaD17S is a codon-optimized delta-17 desaturase
coding region derived from Pythium aphanidermatum (U.S. Pat. No.
7,556,949); and, YICPT1 is a Yarrowia lipolytica diacylglycerol
cholinephosphotransferase coding region (Intl. App. Pub. No. WO
2006/052870).
[0128] Total fatty acid content of the Y4305 cells was 27.5% of dry
cell weight ["TFAs % DCW"], and the lipid profile was as follows,
wherein the concentration of each fatty acid is as a weight percent
of TFAs ["% TFAs"]: 16:0 (palmitate)--2.8, 16:1 (palmitoleic
acid)--0.7, 18:0 (stearic acid)--1.3, 18:1 (oleic acid)--4.9, 18:2
(LA)--17.6, ALA--2.3, EDA--3.4, DGLA--2.0, ARA--0.6, ETA--1.7 and
EPA--53.2.
[0129] Yarrowia lipolytica strain Y4305 F1B1 was derived from Y.
lipolytica strain Y4305. Specifically, strain Y4305 was subjected
to transformation with a dominant, non-antibiotic marker for Y.
lipolytica based on sulfonylurea resistance ["SU.sup.R"]. The
marker gene was a native acetohydroxyacid synthase ("AHAS" or
acetolactate synthase; E.C. 4.1.3.18) that has a single amino acid
change, i.e., W497L, that confers sulfonylurea herbicide resistance
(SEQ ID NO:292 of Intl. App. Pub. No. WO 2006/052870). AHAS is the
first common enzyme in the pathway for the biosynthesis of
branched-chain amino acids and it is the target of the sulfonylurea
and imidazolinone herbicides.
[0130] Random integration of the SU.sup.R marker into Yarrowia
strain Y4305 was used to identify those cells having increased
lipid content when grown under oleaginous conditions relative to
the parent Y4305 strain. Specifically, the mutated AHAS gene
described above was introduced into strain Y4305 cells as a linear
DNA fragment. The AHAS gene integrates randomly throughout the
chromosome at any location that contains a double stranded-break
that is also bound by the Ku enzymes. Non-functional genes or
knockout mutations may be generated when the SU.sup.R marker
fragment integrates within the coding region of a gene. Every gene
is a potential target fordown-regulation. Thus, a random
integration library in Yarrowia Y4305 cells was made and SU.sup.R
mutant cells were identified. Strains were isolated and evaluated
based on DCW (g/L), FAMEs % DCW, EPA % TFAs and EPA % DCW.
[0131] Strain Y4305 F1B1 had 6.9 g/L DCW, 27.9 TFAs % DCW, 53.1 EPA
% TFAs, and 14.8 EPA % DCW as compared to 6.8 g/L DCW, 25.1 TFAs %
DCW, 50.3 EPA % TFAs, and 12.7 EPA % DCW for the control Y4305
strain, when both strains were evaluated in triple flask analysis.
When grown in a two liter fermentation (parameters similar to those
of U.S. Pat. Appl. Pub. No. 2009-009354-A1, Example 10), average
EPA productivity ["EPA % TFAs"] for strain Y4305 was 50-56, as
compared to 50-52 for strain Y4305-F1B1. Average lipid content
["TFAs % DCW"] for strain Y4305 was 20-25, as compared to 28-32 for
strain Y4305-F1B1. Thus, lipid content was increased 29-38% in
strain Y4503-F1B1, with minimal impact upon EPA productivity.
[0132] Yarrowia Biomass Preparation: Inocula were prepared from
frozen cultures of either Yarrowia lipolytica strain Y4305 or
strain Y4305 F1B1 in a shake flask. After an incubation period, the
culture was used to inoculate a seed fermenter. When the seed
culture reached an appropriate target cell density, it was then
used to inoculate a larger fermenter. The fermentation was run as a
2-stage fed-batch process. In the first stage, the yeast were
cultured under conditions that promoted rapid growth to a high cell
density; the culture medium comprised glucose, various nitrogen
sources, trace metals and vitamins. In the second stage, the yeast
were starved for nitrogen and continuously fed glucose to promote
lipid and PUFA accumulation. Process variables including
temperature (controlled between 30-32.degree. C.), pH (controlled
between 5-7), dissolved oxygen concentration and glucose
concentration were monitored and controlled per standard operating
conditions to ensure consistent process performance and final PUFA
oil quality.
[0133] One of skill in the art of fermentation will know that
variability will is occur in the oil profile of a specific Yarrowia
strain, depending on the fermentation run itself, media conditions,
process parameters, scale-up, etc., as well as the particular
time-point in which the culture is sampled (see, e.g., U.S. Pat.
Appl. Pub. No. 2009-0093543-A1).
[0134] Antioxidants were optionally added to the fermentation broth
prior to processing to ensure the oxidative stability of the EPA
oil. After fermentation, the yeast biomass was dewatered and washed
to remove salts and residual medium, and to minimize lipase
activity. Prior to drum drying, ethoxyquin (600 ppm) was added to
the biomass, Then, the biomass was drum dried (typically with 80
psig steam), to reduce the moisture content to less than 5% to
ensure oil stability during short term storage and transportation.
The drum dried biomass was in the form of flakes.
[0135] Extrusion Of Yarrowia Biomass Flakes: Dried biomass flakes
were fed into an extruder, preferably a twin screw extruder with a
length suitable for accomplishing the operations described below,
normally having a length to diameter ["L/D"] ratio between 21-39.
The first section of the extruder was used to feed and transport
the biomass. The following section served as a compaction zone
designed to compact the biomass using bushing elements with
progressively shorter pitch length. After the compaction zone, a
compression zone followed, which served to impart most of the
mechanical energy required for cell disruption. This zone was
created using flow restriction, either in the form of reverse screw
elements or kneading elements. Finally, the disrupted biomass was
discharged through the last barrel which is open at the end, thus
producing no backpressure in the extruder.
[0136] Feed Formulation: The extruded biomass was then formulated
with other feed ingredients (infra) and extruded into pellets using
a 4.5 mm die opening, giving approximately 5.5 mm pellets after
expansion. Yttrium oxide [Y.sub.2O.sub.3] (100 ppm) was added to
all diets as an inert marker for digestibility determination.
Vegetable oil was added post-extrusion to the pellets in accordance
with the diet composition.
Example 1
Oil Composition Of Yarrowia lipolytica Strain Y4305 F1B1 Biomass in
Comparison to Fishmeal, Fish Oil and Rapeseed Oil
[0137] Yarrowia lipolytica strain Y4305 F1B1 biomass was prepared
and made into flakes, as described in General Methods. Oil was
extracted from the whole dried flakes by placing 7 g of dried
flakes and 20 mL of hexane in a 35 mL steel cylinder. Three steel
ball bearings (0.5 cm diameter) were then added to the cylinder and
the cylinder was placed on a vibratory shaker. After 1 hr of
vigorous shaking, the disrupted biomass was allowed to settle and
the solution of oil in hexane was poured off to yield a clear
yellow liquid. This liquid was then poured into a separate tube and
subjected to a nitrogen stream to evaporate the hexane, thereby
leaving the oil phase in the tube. It was determined that about 34%
of the biomass was oil. The composition of the oil was analyzed by
GC, as described in General Methods.
[0138] In addition, the fatty acid composition of fish meal oil,
fish oil and rapeseed oil was similarly analyzed by GC.
[0139] Lipids were extracted as described in General Methods
above.
[0140] A comparison of fatty acids present in the Yarrowia Y4305
F1B1 biomass, fish meal, fish oil, and rapeseed oil is shown in
Table 3. The concentration of each fatty acid is presented as a
weight percent of total fatty acids ["% TFAs"]. EPA is identified
as 20:5, n-3, while DHA is identified as 22:6, n-3.
TABLE-US-00003 TABLE 3 Lipid Composition Of Various Oils Fatty Acid
Fish Yarrowia Common meal Fish Rapeseed Y4305 Fatty acid Name oil
oil oil F1B1 oil C14:0 Myristic 3.7 6.8 0.1 0.1 acid C16:0 Palmitic
10.8 10.5 4.4 2.8 Acid C17:0 -- nd nd nd 0.3 C18:0 Stearic 1.7 1.1
1.8 2.5 acid C20:0 -- 0.1 0.1 0.6 0.8 C22:0 -- <0.1 0.1 0.3 1.1
C24:0 -- nd nd nd 0.6 C16:1, n-7 -- 3.2 4.4 0.2 0.5 C18:1, n-9 --
nd nd nd 4.7 C18:1, n-7 -- nd nd nd 0.4 C18:1, (n-9) + -- 9.4 11.9
59.1 nd (n-7) + (n-5) C20:1, (n-9) + -- 7.6 13.9 1.7 nd (n-7)
C22:1, (n-11) + -- 9.4 20.6 0.9 nd (n-9) + (n-7) C24:1, n-9 -- 0.8
0.9 0.1 nd C16:2, n-4 -- 0.3 0.3 <0.1 nd C16:3, n-4 -- 0.3 0.2
<0.1 nd C16:4, n-1 -- 0.1 0.1 <0.1 nd C18:2, n-6 LA 1.1 1.1
19.3 20.3 C18:3, n-6 GLA 0.1 0.1 <0.1 1.0 C18:3, n-4 -- nd nd nd
0.2 C20:2, n-6 EDA 0.2 0.2 0.1 3.2 C20:3, n-6 DGLA 0.1 0.1 <0.1
1.9 C20:4, n-6 ARA 0.6 0.3 <0.1 0.5 C22:4, n-6 DTA <0.1
<0.1 <0.1 nd C18:3, n-3 ALA 0.7 0.8 8.4 3.4 C18:4, n-3 STA 2
1.9 <0.1 nd C20:1, n-9 -- nd nd nd 0.2 C20:1, n-7 -- nd nd nd
0.6 C20:3, n-3 ETrA 0.1 0.1 <0.1 0.8 C20:3, n-9 -- nd nd nd 0.3
C20:4, n-3 ETA 0.5 0.5 <0.1 0.0 C20:5, n-3 EPA 7.4 5.2 <0.1
46.8 C21:5, n-3 -- 0.3 0.3 <0.1 nd C22:1, n-7 -- nd nd nd 2.0
C22:1, n-11 -- nd nd nd 0.5 C22:5, n-3 DPA 0.6 0.6 <0.1 2.3
C22:6, n-3 DHA 10.6 5.7 <0.1 nd *nd = not detected
[0141] The EPA:DHA ratios for the fishmeal and fish oil samples
were calculated to be 0.7 and 0.9, respectively. In rapeseed oil,
the ratio of EPA and DHA was not determined since EPA and DHA
levels were below detection limits of the analysis. In the Yarrowia
Y4305 F1B1 oil, EPA was very high at 46.8% of total fatty acids,
while DHA was not detected.
[0142] EPA was determined to be about 15% of the Yarrowia Y4305
F1B1 biomass, since EPA constituted 46.8% of the TFAs and fatty
acids (i.e., oil) constituted about 34% of the biomass. Thus, 20%
of Yarrowia Y4305 F1B1 biomass in an aquaculture feed composition
formulation would provide about 3% of EPA by weight in the
aquaculture feed composition.
Example 2
Comparison of a Standard Aquaculture Feed Formulation to an
Aquaculture Feed Formulation Including Yarrowia lipolytica Y4305
F1B1 Biomass
[0143] A standard aquaculture feed formulation was compared to an
aquaculture feed formulation containing Yarrowia Y4305 F1B1
biomass.
[0144] The Yarrowia Y4305 F1B1 biomass-containing aquaculture feed
was formulated using extruded Yarrowia Y4305 F1B1 biomass, prepared
as described in the General Methods (supra). Specifically, a
portion of the fish oil that is typically present in a standard
fish aquaculture feed formulation was replaced with a combination
of Yarrowia Y4305 F1B1 biomass and soybean oil. The prepared
Yarrowia Y4305 F1B1 biomass, which contained about 34% oil (Example
1), was included as 20% of the total feed on a weight basis.
Soybean oil is devoid of EPA and DHA. Fishmeal included in the
aquaculture feed formulation was expected to contribute some EPA
and DHA. Other standard industry ingredients that provide
nutritional benefit in terms of protein, amino acids, fat,
carbohydrate, minerals, energy and astaxanthin were added.
Components of the Yarrowia Y4305 F1B1 biomass-containing
aquaculture feed and the standard aquaculture feed ("control") are
given in Table 4.
[0145] The standard aquaculture feed and Yarrowia Y4305 F1B1
biomass-containing aquaculture feed were produced by extrusion
using 4.5 mm die opening, giving approximately 5.5 mm pellets after
expansion. All aquaculture feed contained 100 ppm Y.sub.2O.sub.3 as
an inert marker for digestibility determination.
[0146] Aquaculture feed samples were analysed for dry matter ["DM"]
(heated at 105.degree. C., until weight was constant), crude
protein (N.times.6.25, Kjeltech Auto System, Tecator, Hoganas,
Sweden), ash (heated at 550.degree. C., until weight was constant),
energy (adiabatic bomb calorimetry) and astaxanthin (as described
by Schierle and Hardi, "Analytical Methods for Vitamins and
Carotenoids in Feeds" In: Hoffmann, Keller, Schierle, Schuep, Eds.
(1994)) (Table 4).
[0147] Additionally, aquaculture feed samples were analysed for
lipids (Soxtec System HT 6 and Soxtec System 1047 Hydrolyzing Unit;
Tecator, Hoganas, Sweden) (Table 4). In addition to the Soxtec
lipid extraction, lipids were extracted by the Folch method (supra)
and fatty acid compositions were analysed by GC. The fatty acid
profiles of the aquaculture feed samples, wherein the concentration
of each fatty acid is presented as a weight percent of total fatty
acids ["% TFAs"], is shown in Table 5. EPA is identified as 20:5,
n-3, while DHA is identified as 22:6, n-3.
[0148] The aquaculture feed samples were also subjected to a water
stability test, using a reduced methodology of the test as
described by G. Baeverfjord et al. (Aquaculture, 261(4):1335-1345
(2006)). Duplicate samples of each diet (10 g each) were placed in
custom made steel-mesh buckets placed inside glass beakers filled
with 300 mL distilled water. The beakers were shaken (100/min) in a
thermostat-controlled water bath (23.degree. C.) for 120 min, and
the remaining amount of dry matter was determined (Table 4).
TABLE-US-00004 TABLE 4 Components And Chemical Compositions In A
Standard Aquaculture Feed Formulation And In Aquaculture Feed
Formulation Including Yarrowia Y4305 F1B1 Biomass Yarrowia Y4305
Component, % Standard Feed F1B1 Feed Fish meal 20.2 20.2 Sunflower
meal, extracted 11.7 3.6 Hydrolyzed feather meal 11.0 13.0 Corn
gluten 9.0 8.9 Yarrowia Y4305 F1B1 biomass 0 20.0 Fish oil 26.0 0
Soybean oil 0 21.0 Soybean meal 4.0 2.0 Wheat 13.5 6.7 Monocalcium
phosphate 1.4 1.4 Vitamin mix 2.0 2.0 Mineral mix 0.4 0.4 L-Lysine
HCl 0.5 0.5 DL-Methionine 0.2 0.2 Carophyll Pink (10% 0.055 0.055
astaxanthin) Yttrium oxide 0.01 0.01 Chemical composition, % Dry
matter 93.6 94.1 Crude fat* 31.1 31.3 Crude protein, N .times. 6.25
37.5 38.7 Ash 5.2 5.9 Energy, MJ/kg 24.5 24.7 Astaxanthin, mg/kg
54.2 58.6 Yttrium, % 0.010 0.010 Minerals P, mg/kg 10471 10775 Ca,
mg/kg 8169 8349 Na, mg/kg 2977 2999 Mg, mg/kg 2519 2048 Zn, mg/kg
160 149 Fe, mg/kg 195 201 Cu, mg/kg 13 12 *See Table 5 for lipid
composition of crude fat.
TABLE-US-00005 TABLE 5 Lipid Composition In A Standard Aquaculture
Feed Formulation And In Aquaculture Feed Formulation Including
Yarrowia Y4305 F1B1 Biomass Yarrowia Standard Y4305 Fatty acid Feed
F1B1 Feed 14:0 7.4 0.5 14:1, n-5 0.4 *nd 15:0 0.3 0.1 16:0 12.3
10.0 16:1, n-5 0.1 0.1 16:1, n-7 4.1 0.5 16:1, n-9 0.2 0.1 16:2,
n-6 0.3 0.1 17:0 0.5 0.1 18:0 1.4 3.4 18:1, n-11 0.7 0.1 18:1, n-7
1.6 1.1 18:1, n-9 11.0 17.1 18:2, n-6 4.5 43.8 18:3, n-3 1.0 5.6
18:3, n-4 0.1 0.2 18:3, n-6 0.1 0.1 18:4, n-3 0.2 0.1 20:0 0.2 0.3
20:1, n-11 1.8 0.2 20:1, n-9 14.1 0.8 20:2, n-6 0.2 0.7 20:3, n-3
0.2 0.2 20:3, n-6 0.0 0.4 20:4, n-3 0.0 0.1 20:4, n-6 0.2 0.1 20:5,
n-3 5.1 9.1 22:0 0.1 0.5 22:1, n-11 21.5 1.1 22:1, n-7 0.4 0.4
22:5, n-3 0.6 0.5 22:6, n-3 5.2 1.0 24:0 0.2 0.3 EPA:DHA 0.98:1 9:1
Ratio *nd = not detected.
[0149] Although the EPA:DHA ratio of the aquaculture feed
formulations are dramatically different (i.e., 0.98:1 for the
standard aquaculture feed formation versus 9:1 for the aquaculture
feed formulation including Yarrowia Y4305 F1B1 biomass, wherein the
biomass was included as 20% of the total aquaculture feed on a
weight basis), the concentration of EPA plus DHA as a weight
percent of total fatty acids ["EPA+DHA % TFAs"] in both aquaculture
feed formulations was similar: 10.3 EPA+DHA TFAs for the standard
feed formation versus 10.1 EPA+DHA % TFAs for the aquaculture feed
formulation including Yarrowia Y4305 F1B1 biomass.
[0150] The total amount of EPA plus DHA, measured as a weight
percent of each aquaculture feed formulation (i.e., "EPA+DHA %"),
can also be calculated by multiplying (EPA+DHA % TFAs)*(total fat
in the aquaculture feed formulation). Thus, the standard
aquaculture feed formulation contained 3.19% EPA+DHA (i.e., [10.3
EPA+DHA % TFAs]*0.31), while the aquaculture feed formulation
including Yarrowia Y4305 F1B1 biomass contained 3.13% EPA+DHA
(i.e., [10.1 EPA+DHA % TFAs]*0.31).
Example 3
Comparison of Standard Feed Formulations to Feed Formulations
Including Variable Percentages of Yarrowia lipolytica Y4305
Biomass
[0151] Two different standard aquaculture feed formulations,
comprising rapeseed oil or a combination of rapeseed and fish oil,
were compared to three different aquaculture feed formulations
containing Yarrowia lipolytica Y4305 biomass.
[0152] As described in the General Methods, while Y. lipolytica
strain Y4305 F1B1 (used in Example 2) contains approximately 28-38%
fat (i.e., measured as average lipid content ["TFAs % DCW"]) and
approximately 15% EPA (i.e., measured EPA content as a percent of
the dry cell weight ["EPA % DCW"]), Y. lipolytica strain Y4305
contains approximately 20-28 TFAs % DCW and approximately 13 EPA %
DCW/. Aquaculture feed formulations comprising the Yarrowia Y4305
biomass, as described in the present Example, were therefore
expected to have different compositions than the aquaculture feed
formulations prepared in Example 2, comprising the Yarrowia Y4305
F1B1 biomass. Additionally, the present Example compares
aquaculture feed formulation components and chemical/lipid
compositions when the Yarrowia Y4305 biomass was included as 10%,
20% or 30% of the total aquaculture feed on a weight basis, i.e.,
designated as "Yarrowia Y4305 Feed-10%", "Yarrowia Y4305 Feed-20%"
and "Yarrowia Y4305 Feed-30%".
[0153] Salmon aquaculture feeds commonly contain either 100% fish
oil or mixtures of vegetable oils and fish oils to achieve
sufficient caloric value and total omega-3 fatty acid content in
the feed formulation. Thus, two standard aquaculture feeds
("control") were prepared in the present Example, the first
comprising 100% rapeseed oil and designated as "Standard
Feed-Rapeseed oil", and the second comprising a mixture of rapeseed
oil and fish oil (1.7:1 ratio) and designated as "Standard
Feed-Fish oil".
[0154] In contrast, each of the aquaculture feed formulations
containing Yarrowia lipolytica Y4305 biomass were prepared with a
mixture of rapeseed oil and Yarrowia Y4305 biomass.
[0155] Yarrowia Y4305 biomass-containing aquaculture feeds were
formulated using extruded Yarrowia Y4305 biomass, prepared as
described in the General Methods (supra). As mentioned above, the
prepared Yarrowia Y4305 biomass was included as either 10%, 20% or
30% of the total feed on a weight basis. Rapeseed oil is
effectively devoid of EPA and DHA. Fishmeal included in the
aquaculture feed formulation was expected to contribute some EPA
and DHA. Other standard industry ingredients of commercial fish
aquaculture feeds that provide nutritional benefit in terms of
protein, amino acids, fat, carbohydrate, minerals, energy and
astaxanthin were added, as in Example 2 and the final formulation
was similarly extruded. The other aquaculture feed components were
balanced across the aquaculture feeds in order to provide identical
levels of protein, fat carbohydrate and energy. Components of the
three Yarrowia Y4305 biomass-containing aquaculture feeds and the
two standard aquaculture feeds ("control") are given in Table
6.
[0156] Following extrusion of the two standard aquaculture feeds
and three Yarrowia Y4305 biomass-containing aquaculture feeds,
aquaculture feed samples were analysed for dry matter ["DM"], crude
protein, ash, energy, astaxanthin and lipids (both by Soxhlet lipid
extraction and by the Folch method) and subjected to a water
stability test, according to the methodologies of Example 2. This
data is summarized in Table 6, while the fatty acid profiles of the
feed samples are shown in Table 7. The concentration of each fatty
acid is presented as a weight percent of total fatty acids ["%
TFAs"]; EPA is identified as 20:5, n-3, while DHA is identified as
22:6, n-3.
TABLE-US-00006 TABLE 6 Components And Chemical Compositions In Two
Alternate Standard Aquaculture Feed Formulations And In Three
Alternate Aquaculture Feed Formulations Including Yarrowia Y4305
Biomass Standard Yarrowia Yarrowia Yarrowia Feed- Y4305 Y4305 Y4305
Standard Rapeseed Feed- Feed- Feed- Feed- oil 10% 20% 30% Fish oil
Formulation, % LT fish meal 48.9 46.1 43.2 40.3 48.9 Wheat gluten
10 10 10 10 10 Yarrowia Y4305 0 10 20 30 0 biomass Fish oil 0 0 0 0
7.34 Rapeseed oil 19.9 18.3 16.7 15.1 12.56 Wheat 18.7 13.1 7.6 2.1
18.7 Vitamin mix 2 2 2 2 2 Mineral mix 0.4 0.4 0.4 0.4 0.4
Carophyll Pink 0.055 0.055 0.055 0.055 0.055 (10% astaxanthin)
Yttrium oxide 0.01 0.01 0.01 0.01 0.01 Chemical composition, % Dry
matter 93.6 91.3 92.7 92.8 93.7 Crude fat* 25.3 24.8 24.7 23.8 25.8
Crude protein, 46.5 43.9 45.3 44.9 45.1 N .times. 6.25 Ash 7.9 7.5
7.3 6.9 8.0 Energy, MJ/kg 23.2 22.8 23.1 23.1 23.5 Astaxanthin,
mg/kg 52.7 48.8 49.2 47.5 56.1 Yttrium, mg/kg 98 98 102 99 99
Minerals P, % 1.18 1.12 1.04 1.02 1.16 Ca, % 1.46 1.36 1.28 1.17
1.39 Mg, mg/kg 1839 1784 1597 1852 1818 Na, mg/kg 7214 5412 5468
6033 5892 Fe, mg/kg 108 127 147 144 112 Mn, mg/kg 32 32 30 39 45
Zn, mg/kg 148 143 143 146 160 Cu, mg/kg 9.3 10.0 10.9 11.3 9.8 *See
Table 7 for lipid composition of crude fat.
TABLE-US-00007 TABLE 7 Lipid Composition In Two Alternate Standard
Aquaculture Feed Formulations And In Three Alternate Aquaculture
Feed Formulations Including Yarrowia Y4305 F1B1 Biomass Standard
Yarrowia Yarrowia Yarrowia Feed- Y4305 Y4305 Y4305 Standard
Rapeseed Feed- Feed- Feed- Feed- oil 10% 20% 30% Fish oil Fatty
acid composition, % 12:0 0.1 *nd *nd *nd *nd 14:0 1.0 0.8 0.8 0.8
2.4 14:1, n-5 *nd *nd *nd *nd 0.1 15:0 *nd 0.1 0.1 0.1 0.2 16:0 6.8
6.6 6.9 7.3 8.0 16:1, n-5 0.1 nd 0.1 0.1 0.1 16:1, n-7 1.1 1.0 1.0
1.0 2.0 16:1, n-9 0.1 0.1 *nd 0.1 0.1 16:2, n-3 0.1 0.1 0.1 0.1 0.1
16:3, n-4 0.1 0.1 0.0 0.0 0.1 17:0 0.1 0.1 0.1 0.2 0.2 17:1, n-7
0.1 0.1 0.1 0.1 0.1 18:0 1.9 2.1 2.4 2.7 1.8 18:1, n-11 0.1 0.1 0.1
0.1 0.3 18:1, n-7 2.9 2.7 2.6 2.5 2.6 18:1, n-9 46.7 46.0 43.5 40.6
37.4 18:2, n-6 17.5 18.1 18.2 18.3 13.8 18:3, n-3 7.1 7.1 6.8 6.4
5.5 18:3, n-4 0.1 0.1 0.1 0.1 0.1 18:3, n-6 0.1 0.1 0.1 0.1 0.1
20:0 0.5 0.5 0.6 0.6 0.4 20:1, n-11 0.5 0.5 0.5 0.5 1.0 20:1, n-7
0.1 0.1 0.1 0.1 0.2 20:1, n-9 3.2 2.8 2.7 2.6 5.6 20:2, n-6 0.1 0.3
0.4 0.6 0.2 20:3, n-3 0.1 0.1 0.1 0.1 *nd 20:3, n-6 *nd 0.2 0.4 0.6
*nd 20:4, n-3 0.3 0.2 0.2 0.2 0.6 20:4, n-6 0.1 0.1 0.1 0.2 0.2
20:5, n-3 1.8 3.0 4.7 6.5 3.1 22:0 0.3 0.3 0.3 0.4 0.2 22:1, n-11
2.4 2.0 1.9 1.9 6.9 22:1, n-7 0.1 0.3 0.5 0.7 0.2 22:1, n-9 0.9 0.9
0.8 0.8 1.1 22:4, n-6 0.3 0.2 0.3 0.5 0.1 22:5, n-3 0.2 0.2 0.2 0.3
0.3 22:6, n-3 2.4 2.2 2.1 2.1 3.6 24:1, n-9 *nd 0.3 0.2 0.2 0.4
EPA:DHA 0.75:1 1.36:1 2.23:1 3.1:1 0.86:1 Ratio *nd = not
detected
[0157] As seen in Table 7, the EPA:DHA ratio of the aquaculture
feed formulations are dramatically different. Each of the
aquaculture feed formulations including Yarrowia Y4305 biomass as a
substitute for fish oil had a higher EPA:DHA ratio than either of
the standard aquaculture feeds comprising 100% rapeseed oil or the
mixture of rapeseed oil and fish oil (i.e., 1.36:1, 2.23:1 and
3.1:1, respectively, versus 0.75:1 and 0.86:1, respectively).
Notably, the Yarrowia Y4305 Aquaculture Feed-20% formulation and
the Yarrowia Y4305 Aquaculture Feed-30% formulation both had
EPA:DHA ratios greater than 2:1.
[0158] The EPA+DHA % TFAs in each of the aquaculture feed
formulations was determined, as described in Example 2.
Specifically, the Standard Feed-Rapeseed Oil formulation had 4.2
EPA+DHA % TFAs or 1.06 EPA+DHA % in the feed, while the Standard
Feed-Fish Oil formulation had 6.7 EPA+DHA % TFAs or 1.73 EPA+DHA %
in the feed. The Yarrowia Y4305 Feed-10% formulation had 5.2
EPA+DHA % TFAs or 1.29 EPA+DHA % in the feed, the Yarrowia Y4305
Feed-20% formulation had 6.8 EPA+DHA % TFAs or 1.68 EPA+DHA % in
the feed and the Yarrowia Y4305 Feed-30% formulation had 8.6
EPA+DHA % TFAs or 2.05 EPA+DHA % in the feed.
Example 4
Comparison of EPA:DHA Ratios in Alternate Aquaculture Feed
Formulations Including Variable Percentages of Yarrowia lipolytica
Y4305 F1B1 Biomass
[0159] A multi-variant analysis was performed to analyze the total
EPA content, total DHA content and ratio of EPA:DHA in a variety of
different model aquaculture feed formulations, wherein the
aquaculture feed formulations comprised: a) either anchovy oil or
menhaden oil, included as 0%, 2%, 5%, 10% or 20% of the total feed
on a weight basis; and, b) Yarrowia lipolytica Y4305 F1B1 biomass,
included as 10%, 20% or 30% of the total feed on a weight
basis.
[0160] As previously noted, salmon aquaculture feeds commonly
contain either 100% fish oil or mixtures of vegetable oils and fish
oils to achieve sufficient caloric value and total omega-3 fatty
acid content in the feed formulation. The fish oil can be purified
from a variety of different fish species, such as anchovy, capelin,
menhaden, herring and cod, and each oil has its own unique fatty
acid lipid profile. For example, anchovy oil was assumed herein to
comprise 17 EPA % TFAs and 8.8 DHA % TFAs, producing a EPA:DHA
ratio of 1.93:1. In contrast, menhaden oil was assumed herein to
comprise 11 EPA % TFAs and 9.1 DHA % TFAs, producing a EPA:DHA
ratio of 1.21:1.
[0161] For the purposes of the calculations herein, the Yarrowia
lipolytica to Y4305 F1B1 biomass was assumed to comprise 15 EPA %
DCW, with no DHA, and biomass of strain Y4305 F1B1 typically
contains an average lipid content of about 28-32 TFAs % DCW (see
General Methods). Both the concentration of EPA as a percent of the
total fatty acids ["EPA % TFAs"] and total lipid content ["TFAs %
DCW"] affect the cellular content of is EPA as a percent of the dry
cell weight ["EPA % DCW"]. That is, EPA % DCW is calculated as:
(EPA % TFAs)*(TFAs % DCW)]/100. Based on the assumptions provided
above with respect to TFAs % DCW and EPA % DCW, the EPA % TFAs for
Yarrowia lipolytica Y4305 F1B1 biomass was calculated to be 50 and
DHA % TFAs was zero.
[0162] Finally, it was necessary to calculate the total EPA content
and total DHA content in the fish meal provided in each aquaculture
feed formulation. It was assumed that the aquaculture feed
formulations containing menhaden oil also included menhaden fish
meal, while the aquaculture feed formulations containing anchovy
oil also included anchovy fish meal. The following set of
assumptions were utilized in the EPA and DHA calculations:
For Anchovy Fish Meal:
[0163] 1. Anchovy fish meal will be included in the final
aquaculture feed formulation as 25% of the total feed on a weight
basis; [0164] 2. Anchovy fish meal is assumed to have a total fat
content of 6%; [0165] 3. One-quarter (25%) of the total fat content
is assumed to be EPA and DHA; [0166] 4. For every 100 g of
aquaculture feed formulation produced, 1.5% of the total
aquaculture feed formulation on a weight basis is total fat content
derived from anchovy fish meal (i.e., 0.25*6). [0167] 5. Since 25%
of total fat content derived from anchovy fish meal in the
aquaculture feed formulation is EPA and DHA, it is assumed that
0.375% of the total aquaculture feed formulation on a weight basis
is EPA and DHA derived from the anchovy fish meal. [0168] 6. Of the
Total EPA+DHA in Anchovy oil, 72% is EPA and 28% is DHA. [0169] 7.
Thus, for every 100 g of aquaculture feed formulation produced,
0.27% is EPA derived from the anchovy fish meal (i.e., 0.375%*0.72)
and 0.1% is DHA derived from the anchovy fish meal (i.e.,
0.375%*0.28).
For Menhaden Fish Meal:
[0169] [0170] 1. Menhaden fish meal will be included in the final
aquaculture feed formulation as 25% of the total feed on a weight
basis; [0171] 2. Menhaden fish meal is assumed to have a total fat
content of 6%; [0172] 3. One-fifth (20%) of the total fat content
is assumed to be EPA and DHA; [0173] 4. For every 100 g of
aquaculture feed formulation produced, 1.5% of the total
aquaculture feed formulation on a weight basis is total fat content
derived from menhaden fish meal (i.e., 0.25*6). [0174] 5. Since 20%
of total fat content derived from menhaden fish meal in the feed
formulation is EPA and DHA, it is assumed that 0.30% of the total
aquaculture feed formulation on a' weight basis is EPA and DHA
derived from the menhaden fish meal. [0175] 6. Of the Total EPA+DHA
in Menhaden oil, 55% is EPA and 45% is DHA. [0176] 7. Thus, for
every 100 g of aquaculture feed formulation produced, 0.165% is EPA
derived from the menhaden fish meal (i.e., 0.30%*0.55) and 0.135%
is DHA derived from the menhaden fish meal (i.e., 0.30%*0.45).
[0177] Based on the assumptions above, it was possible to calculate
the total EPA content, total DHA content and ratio of EPA:DHA in
five different aquaculture feed formulations comprising anchovy oil
(included as 0%, 2%, 5%, 10% or 20% of the total feed on a weight
basis) and Yarrowia lipolytica Y4305 F1B1 biomass (included as 10%,
20% or 30% of the total aquaculture feed on a weight basis) (Table
8). Similarly, total EPA content, total DHA content and ratio of
EPA:DHA in five different aquaculture feed formulations comprising
menhaden oil (included as 0%, 2%, 5%, 10% or 20% of the total
aquaculture feed on a weight basis) and Yarrowia lipolytica Y4305
F1B1 biomass (included as 10%, 20% or 30% of the total aquaculture
feed on a weight basis) were calculated (Table 9).
TABLE-US-00008 TABLE 8 EPA And DHA Content In Aquaculture Feed
Formulations Comprising Variable Concentrations Of Yarrowia Y4305
F1B1 Biomass (10%, 20% And 30%) And Variable Concentrations Of
Anchovy Oil (0%, 2%, 5%, 10% And 20%) % Yarrowia* 30 30 30 30 30 20
20 20 20 20 10 10 10 10 10 % EPA in 4.50 4.50 4.50 4.50 4.50 3.00
3.00 3.00 3.00 3.00 1.50 1.50 1.50 1.50 1.50 Yarrowia* % DHA in
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 Yarrowia* % anchovy oil 0.00 2.00 5.00 10.00 20.00 0.00
2.00 5.00 10.00 20.00 0.00 2.00 5.00 10.00 20.00 % EPA in 0.00 0.34
0.84 1.68 3.35 0.00 0.34 0.84 1.68 3.35 0.00 0.34 0.84 1.68 3.35
anchovy oil % DHA in 0.00 0.18 0.45 0.90 1.80 0.00 0.18 0.45 0.90
1.80 0.00 0.18 0.45 0.90 1.80 anchovy oil % Fish meal 25.00 25.00
25.00 25.00 25.00 25.00 25.00 25.00 25.00 25.00 25.00 25.00 25.00
25.00 25.00 % EPA in 0.27 0.27 0.27 0.27 0.27 0.27 0.27 0.27 0.27
0.27 0.27 0.27 0.27 0.27 0.27 Fish meal % DHA in 0.10 0.10 0.10
0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 Fish
meal Total EPA in 4.77 5.11 5.61 6.45 8.12 3.27 3.61 4.11 4.95 6.62
1.77 2.11 2.61 3.45 5.12 Formulation Total DHA in 0.10 0.28 0.55
1.00 1.90 0.10 0.28 0.55 1.00 1.90 0.10 0.28 0.55 1.00 1.90
Formulation Total EPA + 4.87 5.39 6.16 7.45 10.02 3.37 3.89 4.66
5.95 8.52 1.87 2.39 3.16 4.45 7.02 DHA in Formulation EPA:DHA
47.70: 18.25: 10.20: 6.45: 4.27: 32.70: 12.89: 7.47: 4.95: 3.48:
17.70: 7.54: 4.75: 3.45: 2.69: Ratio 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
*Yarrowia refers to Yarrowia lipolytica strain Y4305 F1B1
biomass.
TABLE-US-00009 TABLE 9 EPA And DHA Content In Aquaculture Feed
Formulations Comprising Variable Concentrations Of Yarrowia Y4305
F1B1 Biomass (10%, 20% And 30%) And Variable Concentrations Of
Menhaden Oil (0%, 2%, 5%, 10% And 20%) % Yarrowia* 30 30 30 30 30
20 20 20 20 20 10 10 10 10 10 % EPA in 4.50 4.50 4.50 4.50 4.50
3.00 3.00 3.00 3.00 3.00 1.50 1.50 1.50 1.50 1.50 Yarrowia* % DHA
in 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 Yarrowia* % menhaden oil 0.00 2.00 5.00 10.00 20.00 0.00
2.00 5.00 10.00 20.00 0.00 2.00 5.00 10.00 20.00 % EPA in 0.00 0.22
0.54 1.08 2.16 0.00 0.22 0.54 1.08 2.16 0.00 0.22 0.54 1.08 2.16
menhaden oil % DHA in 0.00 0.18 0.46 0.92 1.84 0.00 0.18 0.46 0.92
1.84 0.00 0.18 0.46 0.92 1.84 menhaden oil % Fish meal 25.00 25.00
25.00 25.00 25.00 25.00 25.00 25.00 25.00 25.00 25.00 25.00 25.00
25.00 25.00 % EPA in 0.17 0.17 0.17 0.17 0.17 0.17 0.17 0.17 0.17
0.17 0.17 0.17 0.17 0.17 0.17 Fish meal % DHA in 0.13 0.13 0.13
0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 Fish
meal Total EPA in 4.67 4.89 5.21 5.75 6.83 3.17 3.39 3.71 4.25 5.33
1.67 1.89 2.21 2.75 3.83 Formulation Total DHA in 0.13 0.31 0.59
1.05 1.97 0.13 0.31 0.59 1.05 1.97 0.13 0.31 0.59 1.05 1.97
Formulation Total EPA + 4.80 5.20 5.80 6.80 8.80 3.30 3.70 4.30
5.30 7.30 1.80 2.20 2.80 3.80 5.80 DHA in Formulation EPA:DHA
35.92: 15.77: 8.83: 5.48: 3.47: 24.38: 10.94: 6.29: 4.05: 2.71:
12.85: 6.10: 3.75: 2.62: 1.94: Ratio 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
*Yarrowia refers to Yarrowia lipolytica strain Y4305 F1B1
biomass.
[0178] EPA:DHA ratios in the aquaculture feed composition that are
greater than 2:1 were obtained for all combinations of fish oil and
Yarrowia lipolytica Y4305 F1B1 biomass, except in the one case of
the aquaculture feed composition containing 20% menhaden oil in
combination with 10% Yarrowia lipolytica Y4305 F1B1 biomass.
Example 5
Aquaculture of Salmon Using a Standard Aquaculture Feed Formulation
and a Feed Formulation Including Yarrowia lipolytica Y4305 F1B1
Biomass
[0179] The efficacies of the aquaculture feed formulations of
Example 2 were compared in the present Example when used in salmon
aquaculture. Specifically, the effects of the standard aquaculture
feed formulation and the aquaculture feed formulation including 20%
Yarrowia Y4305 F1B1 biomass were compared with respect to total
fish biomass, biomass increase, average body weight, individual
weight gain, pigmentation, dry matter content, crude protein
content, total lipid content and fatty acid profile.
[0180] The experiment was carried out in 15 indoor tanks at Nofima
Marine, Sunndalsora, Norway. Each tank (2 m.sup.2 surface area, 0.6
m water depth) was supplied with seawater (i.e., approximately 33
ppt salinity, at ambient temperature) and stocked with 42 Atlantic
salmon (Salmo salar) of the SalmoBreed strain, mean weight
approximately 495 g. Prior to the experiment, the fish had been
stocked in larger groups in 1 m.sup.2 tanks with similar
conditions. The fish were kept under constant photoperiod during
the experimental period.
[0181] Triplicate tanks of fish were fed by automatic feeders,
aiming at an overfeeding of about 20% to allow maximum feed intake
by the fish. The fish were counted and bulk weighed at the start of
the experiment ["Day 0"], and bulk weighed after 4 weeks ["Day 28"]
of feeding the experimental diets. Any dead fish were removed from
the tanks and weighed immediately.
[0182] At the start of the experiment, fillets were sampled from 3
tanks at 10 fish per tank. This analysis was also performed after 8
and 16 weeks ["Day 53" and "Day 112", respectively] (using 8 fish
per tank at each time period). The color was first measured in the
fresh fillets by a Minolta Chromameter, providing L*a*b values
(wherein "L" is a measure of lightness, "a" is a measure of red
color and "b" is a measure of yellow color). The fillets were
frozen for subsequent analyses of carotenoids, as described by
Bjerkeng et al. (Aquaculture, 157(1-2):63-82 (1997)). Fillets were
also analyzed for dry matter content, crude protein content, total
lipid content and fatty acids. Methods for analyses of fillet,
whole body homogenates and faeces were as described in Example 2
for analyses of feeds.
[0183] Additionally, whole fish were sampled (10 fish per tank) at
the start of the experiment, and homogenized pooled samples of fish
were frozen. After 16 weeks an additional 5 fish per tank were
sampled and homogenized pooled samples of fish were frozen. All
whole body homogenates were analyzed for dry matter content, crude
protein content, total lipid content and fatty acids.
[0184] Results of feeding trials are shown below in Table 10 and
Table 11, with all data reported as the mean, plus or minus
standard error of the mean [".+-.S.E.M"]. Specifically, Table 10
shows total fish biomass (at Days 0, 28, 53 and 112), biomass
["BM"] increases (between Days 0-28, Days 29-53 and Days 54-112),
average body weight (at Days 0, 28, 53 and 112) and individual
weight gain (between Days 0-28, Days 29-53 and Days 54-112). No
unusual mortality was observed during the 112 day trial, evidenced
by comparable weight gains (measured as both biomass per tank of
fish and measured as weight per fish) for fish fed either the
standard feed formulation or the feed formulation including 20%
Yarrowia Y4305 F1B1 biomass.
TABLE-US-00010 TABLE 10 Total Tank Biomass And Fish Weight In
Groups Of Fish Fed A Standard Aquaculture Feed Formulation And An
Aquaculture Feed Formulation Including Yarrowia lipolytica Y4305
F1B1 Biomass Yarrowia Standard Feed Y4305 F1B1 Feed Biomass,
kg/tank Day 0 20788 .+-. 19 20801 .+-. 17 Day 28 23240 .+-. 440
24763 .+-. 168 Day 53 27997 .+-. 490 29132 .+-. 392 Day 112 34342
.+-. 839 35078 .+-. 462 BM Increase, 2452 .+-. 445 3963 .+-. 180
0-28 days BM Increase, 4757 .+-. 78 4369 .+-. 225 29-53 days BM
Increase, 11869 .+-. 520 11241 .+-. 194 54-112 days Average body
weight, g Day 0 495.0 .+-. 0.6 495.3 .+-. 0.7 Day 28 553.3 .+-.
10.7 589.7 .+-. 3.8 Day 53 671.7 .+-. 6.4 688.0 .+-. 7.8 Day 112
1021 .+-. 32 1032 .+-. 14 Weight gain, 58.3 .+-. 10.7 94.3 .+-. 4.3
0-28 days Weight gain, 118.3 .+-. 4.7 98.3 .+-. 5.3 29-53 days
Weight gain, 349.0 .+-. 28.6 343.5 .+-. 12.2 54-112 days
[0185] Table 11 reports the overall composition of the sample fish
fillets (in terms of total protein content, dry matter content, fat
content, pigmentation and fatty acid profile), wherein the fillets
were sampled from fish that were fed either the standard
aquaculture feed formulation or the aquaculture feed formulation
including 20% Yarrowia Y4305 F1B1 biomass. All data is with respect
to grams per 100 grams wet weight of the fish fillet. Values are
reported at Day 0 and at Day 112. EPA is identified as 20:5, n-3,
while DHA is identified as 22:6, n-3.
TABLE-US-00011 TABLE 11 Fatty Acid Composition And Carotenoid
Content Of Salmon Fed Either A Standard Aquaculture Feed
Formulation Or An Aquaculture Feed Formulation Including Yarrowia
lipolytica Y4305 F1B1 Biomass Yarrowia Standard Feed: Y4305 F1B1
Feed: Day 0 Day 112 Day 112 Gross Parameters Dry Matter 28.7 .+-.
0.3 29.7 .+-. 0.4 28.9 .+-. 0.1 Protein 21.7 .+-. 0.2 19.5 .+-. 0.3
19.9 .+-. 0.2 Fat 8.1 .+-. 0.8 10.0 .+-. 0.37 8.8 .+-. 0.14
Carotenoid Content (mg/kg) Astaxanthin 0.5 .+-. 0.sup. 1.87 .+-.
0.12 1.05 .+-. 0.08 Idoxanthin 0.2 .+-. 0.03 0.47 .+-. 0.12 0.73
.+-. 0.13 Fatty Acid Composition 14:0 0.33 .+-. 0.03 0.28 .+-. 0.01
0.17 .+-. 0.01 14:1, n-5 0.02 .+-. 0.00 0.01 .+-. 0.001 0.01 .+-.
0.001 15:0 0.03 .+-. 0.00 0.02 .+-. 0.002 0.02 .+-. 0.001 16:0 1.06
.+-. 0.1 1.14 .+-. 0.04 1.00 .+-. 0.01 16:1, n-5 nd 0.01 .+-. 0.0
0.01 .+-. 0.001 16:1, n-7 0.32 .+-. 0.04 0.24 .+-. 0.01 0.16 .+-.
0.008 16:1, n-9 0.03 .+-. 0.01 0.03 .+-. 0.002 0.02 .+-. 0.001
16:3, n-4 0.03 .+-. 0.00 0.02 .+-. 0.001 0.01 .+-. 0.001 17:0 nd
0.02 .+-. 0.001 0.02 .+-. 0.002 17:1, n-7 nd 0.02 .+-. 0.001 0.01
.+-. 0.001 18:0 0.21 .+-. 0.02 0.28 .+-. 0.01 0.28 .+-. 0.004 18:1,
n-11 0.09 .+-. 0.01 0.10 .+-. 0.005 0.05 .+-. 0.01 18:1, n-7 0.21
.+-. 0.02 0.19 .+-. 0.01 0.17 .+-. 0.004 18:1, n-9 1.15 .+-. 0.10
1.45 .+-. 0.04 1.37 .+-. 0.01 18:2, n-6 0.30 .+-. 0.03 1.69 .+-.
0.05 1.99 .+-. 0.08 18:3, n-3 0.13 .+-. 0.01 0.22 .+-. 0.01 0.25
.+-. 0.01 18:3, n-4 0.02 .+-. 0.00 0.01 .+-. 0.001 0.01 .+-. 0.001
18:3, n-6 nd 0.06 .+-. 0.003 0.06 .+-. 0.004 20:0 0.01 .+-. 0.00
0.02 .+-. 0.001 0.02 .+-. 0.001 20:1, n-11 0.14 .+-. 0.01 0.12 .+-.
0.003 0.10 .+-. 0.003 20:1, n-7 nd 0.02 .+-. 0.001 0.01 .+-. 0.001
20:1, n-9 0.46 .+-. 0.04 0.46 .+-. 0.02 0.25 .+-. 0.01 20:2, n-6
0.04 .+-. 0.00 0.10 .+-. 0.01 0.11 .+-. 0.01 20:3, n-3 0.02 .+-.
0.00 0.02 .+-. 0.001 0.02 .+-. 0.002 20:3, n-6 0.02 .+-. 0.00 0.07
.+-. 0.001 0.08 .+-. 0.002 20:4, n-3 0.08 .+-. 0.01 0.08 .+-. 0.004
0.04 .+-. 0.002 20:4, n-6 0.04 .+-. 0.00 0.04 .+-. 0.001 0.04 .+-.
0.001 20:5, n-3 0.39 .+-. 0.04 0.41 .+-. 0.04 0.34 .+-. 0.03 22:1,
n-11 0.52 .+-. 0.05 0.57 .+-. 0.02 0.27 .+-. 0.01 22:1, n-7 0.09
.+-. 0.01 0.07 .+-. 0.004 0.06 .+-. 0.002 22:1, n-9 0.06 .+-. 0.00
0.06 .+-. 0.002 0.03 .+-. 0.001 22:4, n-6 0.03 .+-. 0.00 0.02 .+-.
0.001 0.02 .+-. 0.001 22:5, n-3 0.16 .+-. 0.02 0.15 .+-. 0.01 0.13
.+-. 0.01 22:6, n-3 1.02 .+-. 0.08 0.76 .+-. 0.03 0.63 .+-. 0.03
24:0 0.01 .+-. 0.01 0.02 .+-. 0.002 0.02 .+-. 0.001 24:1, n-9 0.05
.+-. 0.01 0.04 .+-. 0.002 0.03 .+-. 0.001 EPA + DHA 1.41 .+-. 0.12
1.20 .+-. 0.05 1.00 .+-. 0.02 Sum of n-3 1.82 .+-. 0.16 1.53 .+-.
0.07 1.41 .+-. 0.03 Sum of n-6 0.45 .+-. 0.04 1.23 .+-. 0.04 2.26
.+-. 0.07 Saturated 1.67 .+-. 0.16 1.79 .+-. 0.06 1.52 .+-. 0.02
fatty acids *nd = not detected
[0186] The gross parameters of protein, dry matter, and fat were
very comparable between fish fed the two aquaculture feed
formulations. Astaxanthin was slightly less in fish fed the
aquaculture feed formulation including 20% Yarrowia Y4305 F1B1
biomass.
[0187] With respect to fatty acids, the dominant fatty acids are
identified in bold font in Table 11. The sum of EPA plus DHA
["EPA+DHA"] in the fish at 112 days was similar in fish fed the
standard feed formulation and in fish fed the feed formulation
including 20% Yarrowia Y4305 F1B1 biomass at (i.e., 1.2 g/100 g and
1 g/100 g, respectively).
[0188] Overall, the data suggest that the EPA available in the
Yarrowia Y4305 F1B1 biomass is being adsorbed by the fish and
converted to DHA. This demonstrates that Yarrowia Y4305 F1B1
biomass can be used in place of fish oil in aquaculture feed
formulations for salmon with minimal impact on the health and
growth of the cultured animal.
[0189] Finally, it is noted that the level of 18:2, n-6 (linoleic
acid) in the Yarrowia Y4305 F1B1 biomass results in a significantly
higher total omega-6 content ["Sum of n-6"] in fish fed the feed
formulation including 20% Yarrowia Y4305 F1B1 biomass, as opposed
to in fish fed the standard aquaculture feed formulation. In
commercial practice, fish oil is typically blended with vegetable
oils (e.g., soybean oil or rapeseed oil), which also have higher
levels of 18:2, n-6. Thus, it is anticipated that a less
significant difference would be noted in the 18:2, n-6 content in
fish fed a commercial feed containing soybean or rapeseed oil as
opposed to in fish fed the aquaculture feed formulation including
20% Yarrowia Y4305 F1B1 biomass.
[0190] Based on the results herein, wherein Yarrowia Y4305 F1B1
biomass was successfully used in place of fish oil in aquaculture
feed formulations for salmon, and the calculations set forth in
Example 4, one of skill in the art could readily determine the
appropriate amount of Yarrowia Y4305 biomass or Yarrowia Y4305 F1B1
biomass to be included in various other aquaculture feed
formulations suitable for culture of other fin fish species. The
Yarrowia Y4305 or Y4305 F1B1 biomass could be used to reduce or
replace the total fish oil content in any desired aquaculture feed
formulation. If all other components of the aquaculture feed
formulation containing the Yarrowia Y4305 or Y4305 F1B1 biomass
were comparable to those of the standard feed formulation for a
particular fin fish (i.e., in terms of nutritional benefit,
digestability, palatability, etc.), with the exception of the
Yarrowia Y4305 or Y4305 F1B1 biomass, one of skill in the art would
predict that the modified aquaculture feed formulations containing
the Yarrowia Y4305 or Y4305 F1B1 biomass would be suitable for the
health and growth of the fin fish.
Example 6
Alternate Strains of Yarrowia lipolytica Suitable for Aquaculture
Feed Formulations
[0191] The purpose of this Example is to provide alternate
microbial biomass that could be used as a source of EPA and
optionally DHA, for incorporation into an aquaculture feed
formulation that provides a ratio of concentration of EPA to
concentration of DHA which is greater than 2:1 based on the
individual concentrations of EPA and DHA, each measured as a weight
percent of total fatty acids in the aquaculture feed formulation.
One skilled in the art of aquaculture feed formulation would
readily be able to determine the appropriate amount of biomass (or,
e.g., biomass and oil supplement) to include in the aquaculture
feed formulation, to achieve the desired level of EPA and,
optionally, DHA.
[0192] Although Examples 1-5 demonstrate production and use of
aquaculture feed formulations including Yarrowia lipolytica Y4305
and Yarrowia lipolytica Y4305 F1B1 biomass, the present disclosure
is by no means limited to aquaculture feed formulations comprising
this particular biomass. Numerous other species and strains of
oleaginous yeast genetically engineered for production of .omega.-3
PUFAs are suitable sources of micobial oils comprising EPA. As an
example, one is referred to the representative strains of the
oleaginous yeast Yarrowia lipolytica described in Table 12. These
include the following strains that have been deposited with the
ATCC: Y. lipolytica strain Y2096 (producing EPA; ATCC Accession No.
PTA-7184); Y. lipolytica strain Y2201 (producing EPA; ATCC
Accession No. PTA-7185); Y. lipolytica strain Y3000 (producing DHA;
ATCC Accession No. PTA-7187); Y. lipolytica strain Y4128 (producing
EPA; ATCC Accession No. PTA-8614); Y. lipolytica strain Y4127
(producing EPA; ATCC Accession No. PTA-8802).
[0193] Additionally, Y. lipolytica strain Y8406 (producing EPA;
ATCC Accession No. PTA-10025), Y. lipolytica strain Y8412
(producing EPA; ATCC Accession No. PTA-10026) and Y. lipolytica
strain Y8259 (producing EPA; ATCC Accession No. PTA-10027) are
described in U.S. patent application Ser. No. 12/814,815, filed
Jun. 14, 2010 [E.I. duPont de Nemours & Co., Inc., Attorney
Docket No. "CL4674USNA", hereby incorporated herein by
reference].
[0194] Thus, for example, Table 12 shows microbial hosts producing
from 4.7% to 61.8% EPA of total fatty acids, and optionally, 5.6%
DHA of total fatty acids.
TABLE-US-00012 TABLE 12 Lipid Profiles of Representative Yarrowia
lipolytica Strains Engineered to Produce .omega.-3/.omega.-6 PUFAs
ATCC Fatty Acid Content (As A Percent [%] of Total Fatty Acids)
TFAs Deposit 18:3 20:2 DPAn- % Strain Reference No. 16:0 16:1 18:0
18:1 18:2 (ALA) GLA (EDA) DGLA ARA ETA EPA 3 DHA DCW EU U.S. Pat.
-- 19 10.3 2.3 15.8 12 0 18.7 -- 5.7 0.2 3 10.3 -- -- 36 Y2072
Appl. Pub. -- 7.6 4.1 2.2 16.8 13.9 0 27.8 -- 3.7 1.7 2.2 15 -- --
-- No. 2006- 0115881-A1 Y2102 -- 9 3 3.5 5.6 18.6 0 29.6 -- 3.8 2.8
2.3 18.4 -- -- -- Y2088 -- 17 4.5 3 2.5 10 0 20 -- 3 2.8 1.7 20 --
-- -- Y2089 -- 7.9 3.4 2.5 9.9 14.3 0 37.5 -- 2.5 1.8 1.6 17.6 --
-- -- Y2095 -- 13 0 2.6 5.1 16 0 29.1 -- 3.1 1.9 2.7 19.3 -- -- --
Y2090 -- 6 1 6.1 7.7 12.6 0 26.4 -- 6.7 2.4 3.6 26.6 -- -- 22.9
Y2096 PTA- 8.1 1 6.3 8.5 11.5 0 25 -- 5.8 2.1 2.5 28.1 -- -- 20.8
7184 Y2201 PTA- 11 16.1 0.7 18.4 27 0 -- 3.3 3.3 1 3.8 9 -- -- --
7185 Y3000 U.S. Pat. PTA- 5.9 1.2 5.5 7.7 11.7 0 30.1 -- 2.6 1.2
1.2 4.7 18.3 5.6 -- No. 7187 7,550,286 Y4001 U.S. Pat. -- 4.3 4.4
3.9 35.9 23 0 -- 23.8 0 0 0 -- -- -- -- Y4036 Appl. Pub. -- 7.7 3.6
1.1 14.2 32.6 0 -- 15.6 18.2 0 0 -- -- -- -- Y4070 No. 2009- -- 8
5.3 3.5 14.6 42.1 0 -- 6.7 2.4 11.9 -- -- -- -- -- Y4086 0093543-A1
-- 3.3 2.2 4.6 26.3 27.9 6.9 -- 7.6 1 0 2 9.8 -- -- 28.6 Y4128 PTA-
6.6 4 2 8.8 19 2.1 -- 4.1 3.2 0 5.7 42.1 -- -- 18.3 8614 Y4158 --
3.2 1.2 2.7 14.5 30.4 5.3 -- 6.2 3.1 0.3 3.4 20.5 -- -- 27.3 Y4184
-- 3.1 1.5 1.8 8.7 31.5 4.9 -- 5.6 2.9 0.6 2.4 28.9 -- -- 23.9
Y4217 -- 3.9 3.4 1.2 6.2 19 2.7 -- 2.5 1.2 0.2 2.8 48.3 -- -- 20.6
Y4259 -- 4.4 1.4 1.5 3.9 19.7 2.1 -- 3.5 1.9 0.6 1.8 46.1 -- --
23.7 Y4305 -- 2.8 0.7 1.3 4.9 17.6 2.3 -- 3.4 2 0.6 1.7 53.2 -- --
27.5 Y4127 Int'l. App. PTA- 4.1 2.3 2.9 15.4 30.7 8.8 -- 4.5 3.0
3.0 2.8 18.1 -- -- -- Pub. No. 8802 Y4184 WO -- 2.2 1.1 2.6 11.6
29.8 6.6 -- 6.4 2.0 0.4 1.9 28.5 -- -- 24.8 2008/073367 Y8406 U.S.
patent PTA- 2.6 0.5 2.9 5.7 20.3 2.8 -- 2.8 2.1 0.5 2.1 51.2 -- --
30.7 application 10025 Y8412 No. PTA- 2.5 0.4 2.6 4.3 19.0 2.4 --
2.2 2.0 0.5 1.9 55.8 -- -- 27.0 12/814,815 10026 Y8647 -- 1.3 0.2
2.1 4.7 20.3 1.7 -- 3.3 3.6 0.7 3.0 53.6 -- -- 37.6 Y9028 -- 1.3
0.2 2.1 4.4 19.8 1.7 -- 3.2 2.5 0.8 1.9 54.5 -- -- 39.6 Y9481 --
2.5 0.5 3.1 4.7 11.0 0.6 -- 2.6 3.6 0.9 2.1 60.9 -- -- 35.0 Y9502
-- 2.5 0.5 2.9 5.0 12.7 0.9 -- 3.5 3.3 0.8 2.4 57.0 -- -- 37.1
Y8145 -- 4.3 1.7 1.4 4.8 18.6 2.8 -- 2.2 1.5 0.6 1.5 48.5 -- --
23.1 Y8259 PTA- 3.5 1.3 1.3 4.8 16.9 2.3 -- 1.9 1.7 0.6 1.6 53.9 --
-- 20.5 10027 Y8367 -- 3.7 1.2 1.1 3.4 14.2 1.1 -- 1.5 1.7 0.8 1.0
58.3 -- -- 18.4 Y8672 -- 2.3 0.4 2.0 4.0 16.1 1.4 -- 1.8 1.6 0.7
1.1 61.8 -- -- 26.5
* * * * *